Non-racemic hexafluoroleucine, and methods of making and using it

ABSTRACT

One aspect of the invention relates to hexafluoroleucine and congeners thereof, and methods of making the compounds. Another aspect of the nvention relates to the synthesis of protein cores comprising hexafluoroleucine and congeners thereof. Certain peptides comprising hexafluorleucine and congeners thereof have been characterized using comparative biophysical studies. In general, the fluorinated peptides show higher thermal stability and enhanced resistance to chemical denaturation. Further, mixed hydrocarbonfluorocarbon cores self-sort into homogeneous bundles, suggesting new avenues for the design and manipulation of protein-protein interfaces.

This application claims the benefit of priority to Patent CooperationTreaty Application number PCT/US02/05386, filed Feb. 25, 2002; whichclaims the benefit of priority to U.S. Provisional Patent ApplicationSer. No. 60/271,999, filed Feb. 27, 2001; and U.S. Provisional PatentApplication Ser. No. 60/348,091, filed Oct. 29, 2001.

BACKGROUND OF THE INVENTION

Proteins fold to adopt unique three dimensional structures, usually as aresult of multiple non-covalent interactions that contribute to theirconformational stability. Creighton, T. E. Proteins: Structures andMolecular Properties; 2nd ed.; W. H. Freeman: New York, 1993. Removal ofhydrophobic surface area from aqueous solvent plays a dominant role instabilizing protein structures. Tanford, C. Science 1978, 200,1012–1018; and Kauzmann, W. Adv. Protein Chem. 1959, 14, 1–63. Forinstance, a buried leucine or phenylalanine residue can contribute ˜2–5kcal/mol in stability when compared to alanine. Although hydrogen bondsand salt bridges, when present in hydrophobic environments, cancontribute as much as 3 kcal/mol to protein stability, solvent exposedelectrostatic interactions contribute far less, usually 0.5 kcal/mol.Yu, Y. H.; Monera, O. D.; Hodges, R. S.; Privalov, P. L. J. Mol. Biol.1996, 255, 367–372; and Lumb, K. J.; Kim, P. S. Science 1995, 268,436–439. Hydrogen bonds between small polar side chains and backboneamides can be worth 1–2 kcal/mol, as seen in the case of N-terminalhelical caps. Aurora, R.; Rose, G. D. Protein Sci. 1998, 7, 21–38. Theenergetic balance of these intramolecular forces and interactions withthe solvent determines the shape and the stability of the fold.

While electrostatic interactions in designed structures can provideconformational specificity at the expense of thermodynamic stability,hydrophobic interactions afford a very powerful driving force forstabilizing structures. Recent studies have focused on the introductionof non-proteinogenic, fluorine containing amino acids as a means forincreasing hydrophobicity, without significant concurrent alteration ofprotein structure. Bilgicer, B.; Fichera, A.; Kumar, K. J. Am. Chem.Soc. 2001, 123, 4393–4399; and Tang, Y.; Ghirlanda, G.; Vaidehi, N.;Kua, J.; Mainz, D. T.; Goddard, W. A.; DeGrado, W. F.; Tirrell, D. A.Biochemistry 2001, 40, 2790–2796. The estimated average volumes of CH₂and CH₃ groups are 27 and 54 Å³, respectively, as compared to the muchlarger 38 and 92 Å³ for CF₂ and CF₃ groups. Israelachvili, J. N.;Mitchell, D. J.; Ninham, B. W. Biochim. Biophysica Acta 1977, 470,185–201. Given that the hydrophobic effect is roughly proportional tothe solvent exposed surface area, the large size and volume oftrifluoromethyl groups, in combination with the low polarizability offluorine atoms, results in enhanced hydrophobicity. Tanford, C. TheHydrophobic Effect: Formation of Micelles and Biological Membranes; 2ded.; Wiley: New York, 1980. Indeed, partition coefficients point to thesuperior hydrophobicity of CF₃ (Π=1.07) over CH₃ (Π=0.50) groups.Resnati, G. Tetrahedron 1993, 49, 9385–9445. The low polarizability offluorine also results in low cohesive energy densities of liquidfluorocarbons and is manifested in their low propensities forintermolecular interactions. Riess, J. G. Colloid Surf.-A 1994, 84,33–48; and Scott, R. L. J. Am. Chem. Soc. 1948, 70, 4090–4093. Theseunique properties of fluorine simultaneously bestow hydrophobic andlipophobic character to biopolymers with high fluorine content. Marsh,E. N. G. Chem. Biol. 2000, 7, R153–R157.

Introduction of amino acids containing terminal trifluoromethyl groupsat appropriate positions on protein folds increases the thermalstability and enhances resistance to chemical denaturants. Bilgicer, B.;Fichera, A.; Kumar, K. J. Am. Chem. Soc. 2001, 123, 4393–4399; and Tang,Y.; Ghirlanda, G.; Vaidehi, N.; Kua, J.; Mainz, D. T.; Goddard, W. A.;DeGrado, W. F.; Tirrell, D. A. Biochemistry 2001, 40, 2790–2796.Furthermore, specific protein-protein interactions can be programmed bythe use of fluorocarbon and hydrocarbon side chains. Bilgicer, B.; Xing,X.; Kumar, K. J. Am. Chem. Soc. 2001, 123, 11815–11816. Becausespecificity is determined by the thermodynamic stability of all possibleprotein-protein interactions, a detailed fundamental understanding ofthe various combinations is essential.

The so-called “leucine zipper” protein motif, originally discovered inDNA-binding proteins but also found in protein-binding proteins,consists of a set of four or five consecutive leucine residues repeatedevery seven amino acids in the primary sequence of a protein. In ahelical configuration, a protein containing a leucine zipper motifpresents a line of leucines on one side of the helix. With two suchhelixes alongside each other, the arrays of leucines can interdigitatelike a zipper and/or form side-to-side contacts, thus forming a stablelink between the two helices. Moreover, an increase in thehydrophobicity of the leucine sidechains, e.g., by substitution ofhydrogens with fluorines, in a leucine zipper motif should increase thestrength of the zipper.

Selective fluorination of biologically active compounds is oftenaccompanied by dramatic changes in physiological activities. (a) Welch,T.; Eswarakrishnan, S. Fluorine in Bioorganic Chemistry;Wiley-Interscience: New York, 1991 and references cited therein; (b)Fluorine-containing Amino Acids; Kukhar', V. P., Soloshonok, V. A.,Eds.; John Wiley & Sons: Chichester, 1994; (c) Williams, R. M. Synthesisof Optically Active α-Amino Acids, Pergamon Press: Oxford, 1989; (d)Ojima, I.; Kato, K.; Nakahashi, K.; Fuchikami, T.; Fujita, M. J. Org.Chem. 1989, 54, 4511–4522; (e) Tsushima, T.; Kawada, K.; Ishihara, S.;Uchida, N.; Shiratori, O.; Higaki, J.; Hirata, M. Tetrahedron 1988, 44,5375–5387; (f) Weinges, K.; Kromm, E. Liebigs Ann. Chem. 1985, 90–102;(g) Eberle, M. K.; Keese, R.; Stoeckli-Evans, H. Helv. Chim. Acta 1998,81, 182–186; and (h) Tolman, V. Amino Acids 1996, 11, 15–36. Further,fluorinated amino acids have been synthesized and studied as potentialinhibitors of enzymes and as therapeutic agents. Kollonitsch, J.;Patchett A. A.; Marburg, S.; Maycock, A. L.; Perkins, L. M.; Doldouras,G. A.; Duggan, D. E.; Aster, S. D. Nature 1978, 274, 906–908.Trifluoromethyl containing amino acids acting as potentialantimetabolites have also been reported. (a) Walborsky, H. M.; Baum, M.E. J. Am. Chem. Soc. 1958, 80, 187–192; (b) Walborsky, H. M.; Baum, M.;Loncrini, D. F. J. Am. Chem. Soc. 1955, 77, 3637–3640; and (c) Hill, H.M.; Towne, E. B.; Dickey, J. B. J. Am. Chem. Soc. 1950, 72, 3289–3289.

We describe herein inter alia the design, synthesis, thermodynamiccharacterization and programmed self-sorting of peptide systems withorthogonally miscible hydrocarbon and fluorous, i.e., highly fluorinatedcores.

SUMMARY OF THE INVENTION

A novel, short and efficient synthesis of(S)-5,5,5,5′,5′,5′-hexafluoroleucine (6) in greater than 99% ee startingfrom the protected oxazolidine aldehyde 1 is described. The enantiomericexcess of the product was calculated from an NMR analysis of a dipeptideformed by reaction with a protected L-serine derivative. Furthermore, aracemic sample of N-acylated hexafluoroleucine was enzymaticallyresolved by treatment with porcine kidney Acylase I and was found tohave the same optical rotation as the sample of synthetic 6.

The invention also relates to a method for efficient resolution of thefour diastereomers of 4,4,4-trifluorovaline and 5,5,5-trifluoroleucine.Appropriately derivatized trifluoroamino acids were separated by flashcolumn chromatography into two enantiomeric pairs, which were furtherresolved by porcine kidney acylase I to deliver four pure diastereomers.

Another aspect of the present invention relates to the incorporation ofhexafluoroleucine as a hydrophobic core residue in a designedcoiled-coil, and tailored highly specific protein-protein interactionsbased on the substitution of a hydrophobic core of a protein withfluorinated residues. Another aspect of the invention relates to thedesign and manipulation of specific helix-helix interactions within thecontext of the nonpolar environment of membranes.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts helical wheel representation of residues 1–30 of H and Flooking down the superhelical axis from the N-terminus. All seven coreleucines in H were replaced by hexafluoroleucine (L) in F.

FIG. 2 depicts HPLC traces establishing preferential homodimer formationby fluorous and hydrocarbon cores. Preformed disulfide bondedheterodimer HF (20 μM) was incubated in redox buffer (125 μM oxidizedglutathione, 500 μM reduced glutathione, pH 7.50, 100 mM NaCl, 200 mMMOPS). After 200 minutes, only homodimers and mixed disulfides remain.The mixed heterodimer is estimated to be less than 2% of all H— andF-containing peptides at equilibrium. Peaks marked “*1” and “*2” are Hmonomer and F-glutathione mixed disulfide, respectively, and the peakmarked “**” is an impurity. The equilibrium lies firmly in favor of thehomodimers HH and FF. The free energy of specificity for formation ofhomodimers, ΔG_(spec)=−2.1 kcal/mol.

FIG. 3 depicts: [A] circular dichroism spectra of HH (◯) and FF (●)(conditions: [HH]=[FF]=2 μM, pH 7.40, 137 mM NaCl, 2.7 mM KCl, 10 mMPBS, 10° C.); and [B] thermal denaturation profiles of HH (◯), FF (●)and HF (♦) (conditions: [HH]=[FF]=[HF]=2 μM, 5 M Gdn HCl, pH 7.40, 137mM NaCl, 2.7 mM KCl, 10 mM PBS).

FIG. 4 depicts a representative sedimentation equilibrium trace for FFfrom analytical ultracentrifugation (conditions: 15 μM peptide (FF)conc., pH 7.40, 10 mM phosphate (pH 7.40), 137 mM NaCl, 2.7 mM KCl.Centrifugation: 26 000 rpm, 18 hours equilibration time, 10° C.MW_(calc) (for dimer of FF)=18132, MW_(found)=17385).

FIG. 5 depicts a MALDI mass spectrum of purified peptide HH (calcd.=7556.8 [M+], found=7561).

FIG. 6 depicts a MALDI mass spectrum of purified peptide FF (calcd.=9066 [M+], found=9076.3).

FIG. 7 depicts a MALDI mass spectrum of purified peptide HF (calcd.=8310.4 [M⁺], found=8317). The smaller peaks in the spectrum are the M²⁺peak (4159.9), and monomeric H (3783.3) and monomeric F (4537.7)peptides, resulting from the cleavage of the HF disulfide bond duringthe MALDI experiment.

FIG. 8 depicts the synthesis of the homodimer and the heterodimer. [A]Disulfide bonded homodimers HH and FF were synthesized by air oxidationof monomeric peptides in 6 M Gdn HCl. [B] The heterodimer HF wassynthesized by reaction of H with Ellman's reagent (ER), followed byreaction with excess F.

FIG. 9 depicts thermal melting curves for the two homodimers. [A] HHwith increasing concentrations of guanidine hydrochloride; and [B] FFmonitored by the decrease in molar ellipticity at 222 nm. Peptideconcentration=2 μM

FIG. 10 depicts guanidine hydrochloride melting curves for the twoheterodimers. [A] HH (at 74° C.); and [B] FF (at 80° C.). The data yieldan apparent free energy of unfolding: ΔG_(HH)=+3.90 kcal/mol andΔG_(FF)=+16.76 kcal/mol.

FIG. 11 presents graphically the melting temperatures (T_(m)) as afunction of guanidine hydrochloride concentration for HH (◯) and FF (●).At all temperatures, the fluorinated peptide is more stable.

FIG. 12 depicts a method for the optical resolution of trifluoromethylamino acids. The racemic mixture is N-acylated with acetic anhydride(90% yield), followed by enzymatic cleavage to yield the α-S isomer (99%yield). The stereochemistry at the β (trifluorovaline) andγ(trifluoroleucine) carbons is still unresolved. A method for theproduction of the N-t-Boc-protected amino acid is also depicted.

FIG. 13 depicts stereospecific syntheses of trifluoroleucine andtrifluoronorvaline from L-homoserine (1). An asterisk indicatesunresolved stereochemistry.

FIG. 14 depicts stereospecific syntheses of t-Boc-protectedtrifluorovaline, trifluoroisoleucine and hexafluoroleucine fromoxazolidine aldehyde 10, derived from D-serine. [O]=PCC, NaOAc, 4 Åmolecular sieves (yields range from 50–80%); [**] TsOH, MeOH, rt.

FIG. 15 depicts separation of diastereomeric alcohols 16 by flash columnchromatography, followed by oxidation to give enantiomerically puretrifluorovalines. Comparison of the ¹H and ¹⁹F NMR spectra of dipeptide21 to the corresponding dipeptide obtained from a mixture of (2S,3S)-and (2S,3R)-trifluorovaline shows that there has been no detectableracemization of 21.

DETAILED DESCRIPTION OF THE INVENTION

General Synthesis of Trifluoromethyl Analogs of Leucine, Isoleucine,Valine and Norvaline

We have invented methods to synthesize t-Boc protected trifluoroleucine,trifluorovaline, trifluoroisoleucine, hexafluoroleucine, andtrifluoronorvaline, e.g., with α-S stereochemistry. Xing, X.; Fichera,A.; Kumar, K. “A novel synthesis of enantiomerically pure5,5,5,5′,5′,5′-hexafluoroleucine.” Org. Lett. 2001, 3, 1285–1286. Theseare derived from L-homoserine (FIG. 13) or D-serine (FIG. 14). Anefficient synthesis of trifluoromethionine has been disclosed. Dannley,R. L.; Taborsky, R. G. “Synthesis of DL-S-trifluoromethylhomocysteine(trifluoromethylmethionine).” J. Org. Chem. 1957, 10, 1275–76; andDuewel, H.; Daub, E.; Robinson, V.; Honek, J. F. “Incorporation oftrifluoromethionine into a phage lysozyrne: Implications and a newmarker for use in protein F-19 NMR.” Biochemistry 1997, 36, 3404–3416.

Suitably protected L-homoserine was oxidized to the correspondingaldehyde 2 followed by the fluoride-induced transfer of trifluoromethylgroup from (trifluoromethyl)trimethylsilane. The resulting secondaryfluoro alcohol 3 was oxidized with PCC in 89% yield and then subjectedto Wittig olefination and catalytic hydrogenation to yieldBoc-5,5,5-α-S-trifluorolecuine (6). The stereochemistry at theγ-position is 60% S and 40% R after the reduction reaction, and >99.5% Sat the C_(α)position (ratios were determined by chiral HPLC). Thediastereomerically pure compounds are obtained by reduction to thealcohol followed by chromatographic separation. Alcohol 3 wasdeoxygenated via homolytic reductive cleavage of its thionocarbonateintermediate (7), followed by catalytic reduction to remove the benzylprotecting functionality to yield Boc-5,5,5-α-S-trifluoronorvaline (8).

To install fluorinated side chains on other amino acids that are usuallyfound in hydrophobic cores, we started with the oxazolidine aldehyde 10(Garner aldehyde), available from D-serine in four steps (FIG. 14) whichserves as a chiral non racemic synthon. Campbell, A. D.; Raynham, T. M.;Taylor, R. J. K. “A simplified route to the (R)-Garner aldehyde and(S)-vinyl glycinol.” Synthesis 1998, 1707–1709; Garner, P.; Park, J. M.“The Synthesis and Configurational Stability of Differentially ProtectedBeta-Hydroxy-Alpha-Amino Aldehydes.” J. Org. Chem. 1987, 52, 2361–2364;and Angrick, M. “Note On the Preparation of N-SubstitutedAminoglyceraldehydes.” Mon. Chem. 1985, 116, 645–649. At this stage,trifluoromethyl and pentafluoroethyl groups were introduced usingmethodology similar to that described earlier. The secondary alcoholswere then oxidized to the corresponding ketones in good yield using PCC.The trifluoromethyl ketone 14 was further subjected to a Wittigolefination to yield alkene 15, which after catalytic hydrogenation andoxidation gave Boc-4,4,4-α-S-trifluorovaline (17). The pentafluoroethylketone 18 can be subjected to olefination under similar conditionsfollowed by hydrogenation and oxidation to deliverBoc-5,5,5-trifluoroisoleucine (20). Aldehyde 10 is directly convertedinto the hexafluoro olefin 12 using the phosphonium analog ofMiddleton's phosphorane generated in situ fromtetrakis(trifluoromethyl)-1,3-dithietane and triphenylphosphine.Catalytic hydrogenation was then used to unmask the alcohol andsimultaneously reduce the alkene. The resulting alcohol was thenoxidized using PCC to yield Boc-5,5,5,5′,5′,5′-α-S-hexafluoroleucine.

While the C_(α) stereochemistry is rigorously maintained throughout oursynthetic scheme, the amino acids produced in this manner are still amixture of isomers at the β-position in the case of trifluorovaline andtrifluoroisoleucine. We have found that normal phase chromatography ofalcohols 16 and 19 results in clean separation into the (2S,3S) and(2S,3R) components with recoveries in the 95–100% range. Furthermore,under standard peptide coupling conditions, the stereochemical integrityof the alpha carbon is not compromised.

We have also taken advantage of enzymatic resolution of racemic aminoacids with Acylase I and Lipase. Chenault, H. K.; Dahmer, J.;Whitesides, G. M. “Kinetic resolution of unnatural and rarely occurringamino acids: enantioselective hydrolysis of N-acyl amino acids catalyzedby acylase I.” J. Am. Chem. Soc. 1989, 111, 6354–64; and Houng, J.-Y.;Wu, M.-L.; Chen, S.-T. “Kinetic resolution of amino acid esterscatalyzed by lipases.” Chirality 1996, 8, 418–422. Commerciallyavailable 5,5,5-trifluoroleucine and 4,4,4-trifluorovaline wereacetylated with acetic anhydride and resolved (Acylase I) to yield theα-S amino acids (and >99.9% S stereochemistry at C_(α)) in >90% yield.See FIG. 12; Tsushima, T.; Kawada, K.; Ishihara, S.; Uchida, N.;Shiratori, O.; Higaki, J.; Hirata, M. “Fluorine-containing amino acidsand their derivatives. 7. Synthesis and antitumor activity of α- andγ-substituted methotrexate analogs.” Tetrahedron 1988, 44, 5375; Lazar,J.; Sheppard, W. A. “Fluorinated analogs of leucine, methionine, andvaline.” J. Med. Chem. 1968, 11, 138; Watanabe, H.; Hashizume, Y.;Uneyama, K. “Homologation of trifluoroacetimidoyl iodides bypalladium-catalyzed carbonylation. An approach to α-aminoperfluoroalkanoic acids.” Tetrahedron Lett. 1992, 33, 4333; Larsson, U.;Carlson, R.; Leroy, J. “Synthesis of amino acids with modified principalproperties. 1. Amino acids with fluorinated side chains.” Acta Chem.Scand. 1993, 47, 380–90; Ojima, I.; Kato, K.; Nakahashi, K.; Fuchikami,T.; Fujita, M. “New and effective routes to fluoro analogs of aliphaticand aromatic amino acids.” J. Org. Chem. 1989, 54, 4511–22; Tolmann, V.“Syntheses of fluorinated amino acids. From the classical to the modernconcept.” Amino Acids 1996, 11, 15; Zhang, C.; Ludin, C.; Eberle, M. K.;Stoeckli-Evans, H.; Keese, R. “Asymmetric synthesis of(S)-5,5,5,5′,5′,5′-hexafluoroleucine.” Helv. Chim. Acta 1998, 81, 174;Eberle, M. K.; Keese, R.; Stoeckli-Evans, H. “New synthesis andchirality of (−)-4,4,4,4′,4′,4′-hexafluorovaline.” Helv. Chim. Acta 199881, 182; Keese, R.; Hinderling, C. “Efficient synthesis of (S)-methylhexafluorovalinate.” Synthesis 1996, 695; and Weinges, K.; Kromm, E.“Nonproteinogenic amino acids, II. Synthesis and determination of theabsolute configuration of (2S,4S)-(−)- and(2S,4R)-(+)-5,5,5-trifluoroleucine.” Liebigs Ann. Chem. 1985, 90–102.The selectivity of the acylase reaction was determined by chiral HPLC(CROWNPAK(+)-CR column, Daicel Chemical Industries). The trifluoroderivatives were further Boc protected under mild conditions withoutracemization for use in solid phase peptide synthesis (SPPS).Stereochemistry at the β-carbon (trifluorovaline) and γ-carbon(trifluoroleucine) was left unresolved. In contrast, racemichexafluorovaline resisted resolution by either Lipase or Acylase I.

Specific Synthesis of (S)-5,5,5,5,5′, 5′-Hexafluoroleucine

A novel, short and efficient synthesis of(S)-5,5,5,5′,5′,5′-hexafluoroleucine (6) in greater than 99% ee startingfrom the protected oxazolidine aldehyde 1 is described. The enantiomericexcess of the product was calculated from an NMR analysis of a dipeptideformed by reaction with a protected L-serine derivative. Furthermore, aracemic sample of N-acylated hexafluoroleucine was enzymaticallyresolved by treatment with porcine kidney Acylase I and was found tohave the same optical rotation as the sample of synthetic 6.

Herein, we disclose a novel and efficient synthesis of(S)-5,5,5,5′,5′,5′-hexafluoroleucine starting from commericallyavailable D-serine. For synthesis of α-amino acids derived from D-serineusing a serine aldehyde equivalent, see: Blaskovich, M. A.; Lajoie, G.A. J. Am. Chem. Soc. 1993, 115, 5021–5030. While there is one existingreport of the synthesis of racemic hexafluoroleucine (Lazar, J.;Sheppard, W. A. J. Med. Chem. 1968, 11, 138), and another recent reportdetailing the preparation of 6 in 81% ee (Zhang, C.; Ludin, C.; Eberle,M. K.; Stoeckli-Evans, H.; Keese, R. Helv. Chim. Acta 1998, 81,174–181), we have discovered a method to obtain hexafluoroleucine in>99%ee, e.g., for direct use in solid phase peptide synthesis.

Our synthesis commenced from the oxazolidine aldehyde 1 (Garneraldehyde) which served as a chiral, nonracemic synthon. See (a) Garner,P.; Park, J. M. J. Org. Chem. 1987, 52, 2361–2364. (b) Garner, P.; Park,J. M. J. Org. Chem. 1988, 53, 2979–2984. (c) Garner, P.; Park, J. M.;Malecki, E. J. Org. Chem. 1988, 53, 4395–4398. (d) Angrick, M. Montash.Chem. 1985, 116, 645–649. Aldehyde 1 is derived from D-serine and wasobtained using a slight modification of a published procedure and isexceptionally stable towards racemization in subsequent steps. Campbell,A. D.; Raynham, T. M.; Taylor, R. J. K. Synthesis 1998, 1707–1709. In akey step, aldehyde 1 was converted to the bis-trifluoromethyl olefin 2by a Wittig reaction in 92% yield (Scheme 1). See Korhummel, C.; Hanack,M. Chem. Ber. 1989, 122, 2187–2192.

The ylide for this reaction is the phosphonium analog of Middleton'sphosphorane, Middleton, W. J.; Sharkey, W. H. J. Org. Chem. 1965, 30,1384, generated in situ from tetrakis(trifluoromethyl)-1,3-dithietane(Anello, L. G.; Vanderpuy, M. J. Org. Chem. 1982, 47, 377–378), andtriphenyl phosphine. See (a) Burton, D. J.; Yang, Z. Y.; Qiu, W. M.Chem. Rev. 1996, 96, 1641–1715. (b) Dixon, D. A.; Smart, B. E. J. Am.Chem. Soc. 1986, 108, 7172–7177. (c) Burton, D. J.; Inouye, Y.Tetrahedron Lett. 1979, 3397–3400; and (d) Kobayashi, Y.; Nakajima, M.;Nakazawa, M.; Taguchi, T.; Ikekawa, N.; Sai, H.; Tanaka, Y.; Deluca, H.F. Chem. Pharm. Bull. 1988, 36, 4144–4147. The olefin 2 was reduced bycatalytic hydrogenation over Pd/C to give the suitably substitutedoxazolidine 3 in 98% yield. Next, the oxazolidine was subjected to acidcatalyzed ring cleavage unmasking the alcohol 4. Alcohol 4 was oxidizedto the carboxylic acid 5 using pyridinium dichromate and in the finalstep, the t-butyloxycarbonyl group was removed using trifluoroaceticacid to yield the hydrochloride salt of the desired α-amino acid 6.While the last deprotection step was carried out in order to verify theoptical purity of 6, the Boc protected amino acid 5 could be directlyused for solid phase synthesis of peptides.

The optical purity of synthetic 6 was verified in two ways. A racemicsample of 5 (prepared using a different route) and 5 obtained throughthe scheme described here were separately coupled to a protected methylester of L-serine (7), and the resulting dipeptide was analyzed using ¹HNMR spectroscopy.

In the case of the dipeptide obtained from racemic 5, three signalscorresponding to the t-Boc group, the methyl ester and the t-butyl etherwere split into two peaks, presumably due to formation of twodiastereomers; whereas, 5 from the present synthesis yielded a dipeptidewith only one set of signals for the same three sets of protons.Further, racemic 6 was N-acylated and enzymatically resolved usingporcine kidney Acylase I [E.C.N. 3.5.1.14] to yield the α-S isomerexclusively. See (a) Chenault, H. K.; Dahmer, J.; Whitesides, G. M. J.Am. Chem. Soc. 1989, 111, 6354–64; and (b) Fu, S. C. J.; Birnbaum, S. M.J. Am. Chem. Soc. 1953, 75, 918–920. The optical rotation of 6 obtainedin this manner and that of the synthetic sample were identical. Thus,the synthesis proceeds in greater than >99% ee. The NMR data for 6 agreewith those reported previously. Moreover, both the synthetic sample andthe enzyme resolved samples of 6 had [α]²⁶⁰ _(D)=+5.6° (c 1, CH₃OH).Likewise, the construction of 5,5,5,5′,5′,5′-(R)-hexafluoroleucine wasachieved from L-senne.

Resolution of the Diastereomers of 4,4,4-Trifluorovaline and5,5,5-Trifluoroleucine

Reported here is an efficient resolution of the four diastereomers of4,4,4-trifluorovaline (TFV) and 5,5,5-trifluoroleucine (TFL). The methodas outlined in Scheme 1 is simple and practical. Appropriatelyderivatized TFL and TFV could be separated into two enantiomeric pairsby flash column chromatography. Subsequent enzymatic deacylation of theN-acetyl enantiomeric pairs of amino acids with porcine kidney acylase Idelivers all four diastereomers in optically pure form. Chenault, H. K.;Dahmer, J.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 6354–6364.

In the course of our study on the synthesis of enantiomerically pureTFV, we found that Boc-protected 4,4,4-trifluorovalinol α-Sdiastereomers could be easily separated by column chromatography onsilica gel. This finding encouraged us to develop a resolution schemefor racemic TFV and TFL. As shown in Scheme 2, Boc-TFV 1 was firstconverted to Boc-trifluorovalinol 2 via esterification of 1 with methyliodide, followed by reduction of the methyl ester with sodiumborohydride in methanol in 73% overall yield for the two steps. Theracemic mixture of trifluorovalinols was easily separated into the twoenantiomeric pairs 2a [(2S,3S)+(2R,3R)] and 2b [(2S,3R)+(2R,3S)] bycolumn chromatography on silica gel using n-pentane/ethyl ether (1:1) aseluant. Although the methyl esters of Boc-TFV 1 are also separable, theyare not stable toward racemization in the subsequent reduction step.Oxidation of the hydroxyl group of 2a and 2b with PDC in DMF, removal ofthe Boc-protecting group with 30% trifluoroacetic acid in methylenechloride followed by acylation of the free amino group afforded theN-acetyl amino acids 3a and 3b respectively. Finally, enzymaticdeacylation of 3a and 3b with porcine kidney acylase I afforded the fourdiastereomers 4a–d. Only those diastereomers that had an S configurationat C_(α) were deacylated by the enzyme. Removal of the acetyl group fromthe two C_(α)-R diastereomers was realized by refluxing with 3 N HCl.

-   -   Reagents and conditions: (a) NaHCO₃, CH₃I, DMF, rt, 95%; (b)        NaBH₄, CH₃OH, 77%; flash column chromatography, n-pentane/Et₂O        (1:1), silica gel:2 (300:1); (c) PDC, DMF, rt, 65%; (d) 30%        CF₃CO₂H/CH₂Cl₂, 100%; (e) NaOH/H₂O, Ac₂O, 0° C., 95%; (f)        Porcine kidney acylase I, pH 7.50, 25° C., 95%; (g) 3N HCl, 98%.

This strategy was also applied to the resolution of TFL (Scheme 3).Initially, Boc-TFL 5 was also converted to the corresponding alcoholsfollowing the procedure used for Boc-TFV 1, but we found that thetrifluoroleucinols were not separable by column chromatography on silicagel. Interestingly, the methyl esters of 5 were readily separated intotwo pairs 6a and 6b on silica gel using n-pentane/ethyl ether (3:1) aseluant and were stable toward racemization in the reduction step. TheN-acetyl amino acids 7a and 7b were obtained from 6a and 6b respectivelyby straightforward functional group transformations, which includedreduction of the methyl ester group to hydroxyl, oxidation of thehydroxyl to acid, and replacement of the Boc-protecting group with anacetyl group. In the final step, enzymatic deacylation was applied to 7aand 7b to give diastereomerically pure compounds 8a–d.

The purity of the intermediates and the final diastereomers wasascertained using ¹H, ¹³C and ¹⁹F NMR spectroscopy. The ¹⁹F NMRtechnique is particularly useful in this case for purity control due toits high sensitivity and the large chemical shift dispersion observedfor these compounds. The enantiomeric pairs exhibited baseline separated¹⁹F NMR spectra in each case. Contamination by the other enantiomericpair or racemization during chemical transformation could be easilydetected. The optical purity of the products was also verified by NMRanalysis of dipeptides formed by coupling with a side chain protectedmethyl ester of L-serine. Xing, X.; Fichera, A.; Kumar, K. Org. Lett.2001, 3, 1285–1286. The ¹⁹F NMR spectra clearly showed four peaks fordipeptides derived from the racemic mixture, two peaks for dipeptidesderived from enantiomeric pairs, and only one peak for thediastereomerically pure dipeptide.

Programmed Sel-Sorting of Coiled Coils with Leucine andHexafluoroleucine Cores

The coiled coil motif offers an excellent model system to explorespecificity in protein-protein interactions. Lupas, A. Curr. Opin.Struct. Biol. 1997, 7, 388–393; and Lupas, A. Trends Biochem. Sci. 1996,21, 375–382. These protein interaction motifs represent small,synthetically tractable targets for testing hypothetical constructs.Lajmi, A. R.; Lovrencic, M. E.; Wallace, T. R.; Thomlinson, R. R.; Shin,J. A. J. Am. Chem. Soc. 2000, 122, 5638–5639. The α-helical coiled coilis typically composed of a number of parallel or antiparallel α-heliceswrapped around one another with a shallow left-handed superhelicaltwist. Crick, F. H. C. Acta Crystallographica 1953, 6, 689–697. Theycontain a heptad repeat, whose positions are denoted a–g, where the aand d positions are hydrophobic residues that form the interface betweenhelices, and constitute the primary driving force for oligomerization.Additionally, interhelical electrostatic interactions between e and gresidues provide a secondary source of stability. Monera, O. D.; Zhou,N. E.; Kay, C. M.; Hodges, R. S. J. Biol. Chem. 1993, 268, 19218–19227;and Monera, O. D.; Kay, C. M.; Hodges, R. S. Biochemistry 1994, 33,3862–3871. From the crystal structures of 32-residue synthetic coiledcoils, it is estimated that nearly 900 Å² surface area per helix isburied at a dimeric interface and nearly 1640 Å² per helix in atetramer. Harbury, P. B.; Zhang, T.; Kim, P. S.; Alber, T. Science 1993,262, 1401–1407; and O'Shea, E. K.; Klemm, J. D.; Kim, P. S.; Alber, T.Science 1991, 254, 539–544. The importance of hydrophobic surface areafor coiled coil stability has been extensively studied through the useof de novo designed synthetic peptide models. Zhu, B. Y.; Zhou, N. E.;Kay, C. M.; Hodges, R. S. Protein Sci. 1993, 2, 383–394; and Zhu, B. Y.;Zhou, N. E.; Semchuk, P. D.; Kay, C. M.; Hodges, R. S. Int. J. Pept.Protein Res. 1992, 40, 171–179. These interaction surfaces are thereforeideally suited to study the effect of fluorination on the driving forceand specificity.

Peptides were synthesized by the in situ neutralization protocol fort-Boc synthesis on 0.40 mmol NH₂ eq g⁻¹ methylbenzhydrylamine (MBHA)resin. At the end of linear synthesis, the formyl protecting group onthe tryptophan residue was removed by treatment with 1:10 piperidine inDMF solution at 0° C. for 2 hrs. Further treatment with anhydrous HFresulted in the simultaneous removal of all side-chain protecting groupsand cleavage of the peptide chain from the resin. The peptides werepurified on reversed-phase HPLC using a linear gradient of acetonitrilein 0.1% trifluoroacetic acid (TFA)/water. The analytical purity of thepeptides was confirmed by HPLC, amino acid analysis and MALDI massspectrometry.

The disulfide bonded dimers of H (HH), F (FF) and the mixed dimer HFwere synthesized by two different methods. The homodimers HH and FF weresynthesized by overnight air oxidation of the monomeric peptides in 6 Mguanidine hydrochloride (Gdn HCl) at pH 8.50 (50 mM Tris). Theheterodimer HF was synthesized by reaction of H with a large excess ofEllman's reagent (ER, CAS No. 69-78-3) to produce an activated disulfidespecies at pH 7.50, followed by reaction with excess monomeric F at pH5.10. Riddles, P. W.; Blakeley, R. L.; Zemer, B. Methods Enzymol. 1983,91, 49–60. The resulting heterodimer HF was purified by reversed-phaseHPLC.

Peptides H and F are equipped with N-terminal cysteine residues and weredesigned to form parallel homodimeric coiled coil assemblies. Wolf, E.;Kim, P. S.; Berger, B. Protein Sci. 1997, 6, 1179–1189. These peptideshave an identical sequence except that all seven of the core leucineresidues in H have been replaced by 5,5,5,5′,5′,5′-α-S-hexafluoroleucinein F, shielding 28 trifluoromethyl groups from aqueous solvent in thecanonical fluorinated dimer. FIG. 1. Hexafluoroleucine was synthesizedaccording to the procedure described herein. Xing, X.; Fichera, A.;Kumar, K. Org. Lett. 2001, 3, 1285–1286. The peptides were assembled on4-methylbenzhydrylamine (MBHA) resin according to the in situneutralization protocol for t-Boc peptide synthesis, as describedpreviously, and purified by reverse-phase HPLC. Schnolzer, M.; Alewood,P.; Jones, A.; Alewood, D.; Kent, S. B. Int. J. Pept. Protein Res. 1992,40, 180–193. Purity of the peptides was confirmed by analytical HPLC andMALDI mass spectrometry. H and F are designed to form parallel coiledcoil structures due to unfavorable interhelical electrostaticinteractions in the antiparallel arrangements. See Lumb, K. J.; Kim, P.S. Biochemistry 1995, 34, 8642–8648; and Harbury, P. B.; Zhang, T.; Kim,P. S.; Alber, T. Science 1993, 262, 1401–1407. Furthermore, a singlepolar residue, Asn14, which can only hydrogen bond in the parallelarrangement, was incorporated in the hydrophobic core. See Oakley, M.G.; Kim, P. S. Biochemistry 1998, 37, 12603–12610; and McClain, D. L.;Woods, H. L.; Oakley, M. G. J. Am. Chem. Soc. 2001, 123, 3151–3152. Thepeptides were equipped with a Gly-Gly-Cys tripeptide at theNH₂-terminus. The cysteine residue permits redox chemistry in the formof disulfide-thiol equilibrium, and the two glycine residues provide aflexible linker. Disulfide bonded dimers of H (HH) and F (FF) weresynthesized by air oxidation of the monomeric peptides in pH 8.50 Trisbuffer.

The extent of the preference for sorting into homodimeric populationsunder equilibrium conditions was examined by a disulfide exchange assay.See Harbury, P. B.; Kim, P. S.; Alber, T. Nature 1994, 371, 80–83;Oakley, M. G.; Kim, P. S. Biochemistry 1998, 37, 12603–12610; andSaghatelian, A.; Yokobayashi, Y.; Soltani, K.; Ghadiri, M. R. Nature2001, 409, 797–80. Preformed disulfide bonded heterodimer HF wasincubated in a pH 7.50 redox buffer at 20° C., conditions under whichdisulfide exchange is rapid. Aliquots were removed from the reaction atvarious times and quenched with 5% trifluoroacetic acid. The time pointswere then analyzed by analytical reversed-phase HPLC. Relativeconcentrations of the disulfide bonded hetero- and homodimers wereestimated by integration of the area under corresponding peaks at 230nm. Within 30 minutes of the start of the reaction, the heterodimerdisproportionates into the two homodimers HH and FF. Specifically, weobserved about 10% of the H-gluathione and 20% of the F-glutathionedisulfide adducts. Coincidentally, the H-glutathione disulfide co-elutedwith HH. After 200 minutes, only a trace of the heterodimer (˜3%)remains. FIG. 2. Further change in the reaction mixture was not observedeven after 18 hours. Assuming that the glycyl linkers allow thecysteines to exchange randomly under redox buffer conditions, the dataindicate that the homodimers are preferred over the heterodimer by26-fold. In order to establish that the reaction had reachedequilibrium, we placed an equimolar amount of the reduced peptides H andF under similar redox buffer conditions, and monitored the reaction for18 hours. Again, the heterodimer accounted for only 3% of all disulfidebonded species. Unambiguous stepwise synthesis of the heterodimer HFconfirms that the disulfide bond forming chemistry is reversible andunder thermodynamic control, and that there are no kinetic barriers tothe formation of the disulfide bonded heterodimer HF. The heterodimer HFwas synthesized by reaction of H with Ellman's reagent to produce anactivated disulfide species. This mixed disulfide was then reacted withexcess monomeric F to yield HF. See Riddles, P. W.; Blakeley, R. L.;Zemer, B. Methods Enzymol. 1983, 91, 49–60.

Accordingly, peptides H and F are predisposed to form homodimers. SeeOtto, S.; Furlan, R. L. E.; Sanders, J. K. M. J. Am. Chem. Soc. 2000,122, 12063–12064; Hioki, H.; Still, W. C. J. Org. Chem. 1998, 63,904–905; and Rowan, S. J.; Hamilton, D. G.; Brady, P. A.; Sanders, J. K.M. J. Am. Chem. Soc. 1997, 119, 2578–2579. The relative instability ofthe heterodimer and the hyperstability of the fluorinated dimer providethe driving force for preferential homodimer formation. From the peakratios at equilibrium, the free energy of specificity for the formationof homodimers, ΔG_(spec), is calculated to be at least −2.1 kcaumol. SeeExample 11.

TABLE 1 Melting temperatures and solution MWs for disulfide bondeddimers. Peptide T_(m) (° C.)^(a) MW_(app) (no. of helices)^(c) HH 34 7501 ± 38 (2) HF 36  8815 ± 63^(d) (2) FF 82 (45^(b)) 17835 ± 75 (4)^(a)Determined by monitoring the molar ellipticity at 222 nm as afunction of temperature. Conditions: 2 μM peptide conc.; pH 7.40, 5 MGdn HCl, 10 mM PBS. ^(b)In 7 M Gdn HCl, pH 7.40, 10 mM PBS.^(c)Determined by sedimentation equilibrium. Conditions: 15 μM peptideconc., pH 7.40, 10 mM PBS, 10° C. ^(d)Non random residuals.

Circular dichroism spectra of peptides HH, HF and FF revealed the alphahelical character of all three disulfide bonded dimers, showingcharacteristic minima at 208 and 222 nm. FIG. 3[A]. The order ofstability was readily established when melting curves were monitored byCD are compared. All three peptides HF, RH and FF displayed cooperativeunfolding transitions as a function of temperature in the presence ofguanidine hydrochloride (Gdn HCl). The melting temperatures in 5 M GdnHCl of HH (34° C.) and that of HF (36° C.) were similar. In contrast,the fluorinated peptide FF meltrd at an estimated 82° C. under theseconditions. FIG. 3[B]. The fluorinated disulfide bonded dimer displayedremarkable stability, resisting even minimal denaturation at 6 M Gdn HClat room temperature. Even at 7 M Gdn HCl concentration, FF resistedthermal denaturation up to 45° C. Table 1. Thus, the fluorinatedassembly FF is significantly more stable than either the heterodimer HFor the hydrocarbon homodimer HH. A priori, the T_(m) of the heterodimercan be expected to be the average of the T_(m) values of the homodimers(ΔT_(m)=0). The specificity for heterodimer formation can beapproximated by ΔT_(m)=T_(m)(heterodimer HF) −½ [T_(m)(homodimerHH)+T_(m)(homodimer FF)]=−22° C. Differences in ΔT_(m) have been invokedto explain the specificity of the heterodimeric Fos-Jun peptide pair.O'Shea, E. K.; Rutkowski, R.; Kim, P. S. Cell 1992, 68, 69–708. In ourcase, ΔT_(m) is −22° C., i.e. the thermal stability of the heterodimeris appreciably lower than the expected intermediate stability. Thethermodynamic consequence of the relative stability of the fluorinatedpeptide assembly FF and the instability of HF is to shift theequilibrium away from the heterodimer to the homodimers.

Sedimentation equilibrium analysis of the disulfide bonded dimers in the2–15 μM range revealed that HH has an apparent molecular weight of 7501D in solution, consistent with two helices forming the coiled coilstructure. Table 1. In contrast, FF sediments with an apparent molecularweight of 17835 D. This could be due to much larger association constantof FF monomers or due to the larger size of the core formed byhexafluoroleucine forcing it to adopt a coiled coil structure with fourhelices.

In sum, we have demonstrated the incorporation of hexafluoroleucine asthe sole hydrophobic core residue in a designed coiled-coil.Furthermore, this is the first example of a very highly specificprotein-protein interaction based on the substitution of the hydrophobiccore with fluorinated residues. This aspect of the invention relates toa method to design and manipulate specific helix-helix interactionswithin the context of the nonpolar environment of membranes. See Choma,C.; Gratkowski, H.; Lear, J. D.; DeGrado, W. F. Nature Struct. Biol.2000, 7, 161–166; and Zhou, F. X.; Cocco, M. J.; Russ, W. P.; Brunger,A. T.; Engelman, D. M. Nature Struct. Biol. 2000, 7, 154–160.

Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are boron, nitrogen,oxygen, phosphorus, sulfur and selenium.

The term “electron-withdrawing group” is recognized in the art, anddenotes the tendency of a substituent to attract valence electrons fromneighboring atoms, i.e., the substituent is electronegative with respectto neighboring atoms. A quantification of the level ofelectron-withdrawing capability is given by the Hammett sigma (σ)constant. This well known constant is described in many references, forinstance, J. March, Advanced Organic Chemistry, McGraw Hill BookCompany, New York, (1977 edition) pp. 251–259. The Hammett constantvalues are generally negative for electron donating groups (σ[P]=−0.66for NH₂) and positive for electron withdrawing groups (σ[P]=0.78 for anitro group), σ[P] indicating para substitution. Exemplaryelectron-withdrawing groups include nitro, acyl, formyl, sulfonyl,trifluoromethyl, cyano, chloride, and the like. Exemplaryelectron-donating groups include amino, methoxy, and the like.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁–C₃₀ for straight chain, C₃–C₃₀ for branchedchain), and more preferably 20 or fewer. Likewise, preferred cycloalkylshave from 3–10 carbon atoms in their ring structure, and more preferablyhave 5, 6 or 7 carbons in the ring structure.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Preferred alkyl groups are lower alkyls. Inpreferred embodiments, a substituent designated herein as alkyl is alower alkyl.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group).

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The term “aryl” as used herein includes 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“heteroaromatics.” The aromatic ring can be substituted at one or morering positions with such substituents as described above, for example,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN, or the like. The term “aryl” alsoincludes polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings (the ringsare “fused rings”) wherein at least one of the rings is aromatic, e.g.,the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls and/or heterocyclyls.

The terms ortho, meta andpara apply to 1,2-, 1,3- and 1,4-disubstitutedbenzenes, respectively. For example, the names 1,2-dimethylbenzene andortho-dimethylbenzene are synonymous.

The terms “heterocyclyl” or “heterocyclic group” refer to 3- to10-membered ring structures, more preferably 3- to 7-membered rings,whose ring structures include one to four heteroatoms. Heterocycles canalso be polycycles. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine,phenanthroline, phenazine, phenarsazine, phenothiazine, furazan,phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine,piperazine, morpholine, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringcan be substituted at one or more positions with such substituents asdescribed above, as for example, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like.

The terms “polycyclyl” or “polycyclic group” refer to two or more rings(e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Rings that are joined throughnon-adjacent atoms are termed “bridged” rings. Each of the rings of thepolycycle can be substituted with such substituents as described above,as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromaticmoiety, —CF₃, —CN, or the like.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formula:

wherein R₉, R₁₀ and R′₁₀ each independently represent a group permittedby the rules of valence.

The term “acylamino” is art-recognized and refers to a moiety that canbe represented by the general formula:

wherein R₉ is as defined above, and R′₁₁ represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as defined above.

The term “amido” is art recognized as an amino-substituted carbonyl andincludes a moiety that can be represented by the general formula:

wherein R₉, R₁₀ are as defined above. Preferred embodiments of the amidewill not include imides which may be unstable.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S—(CH₂)_(m)—R₈, wherein m and R₈ are defined above.Representative alkylthio groups include methylthio, ethyl thio, and thelike.

The term “carbonyl” is art recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R₁₁represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R₈ or apharmaceutically acceptable salt, R′ ₁₁ represents a hydrogen, an alkyl,an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as defined above. WhereX is an oxygen and R₁₁ or R′₁₁ is not hydrogen, the formula representsan “ester”. Where X is an oxygen, and R₁₁ is as defined above, themoiety is referred to herein as a carboxyl group, and particularly whenR₁₁ is a hydrogen, the formula represents a “carboxylic acid”. Where Xis an oxygen, and R′₁₁ is hydrogen, the formula represents a “formate”.In general, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiolcarbonyl” group. Where X is asulfur and R₁₁ or R′₁₁ is not hydrogen, the formula represents a“thiolester.” Where X is a sulfur and R₁₁ is hydrogen, the formularepresents a “thiolcarboxylic acid.” Where X is a sulfur and R₁₁′ ishydrogen, the formula represents a “thiolformate.” On the other hand,where X is a bond, and R₁₁ is not hydrogen, the above formula representsa “ketone” group. Where X is a bond, and R₁₁ is hydrogen, the aboveformula represents an “aldehyde” group.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynl, —O—(CH₂)_(m)—R₈,where m and R₈ are described above.

The term “sulfonate” is art recognized and includes a moi ty that can berepresented by the general formula:

in which R₄₁ is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized andrefer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl,and nonafluorobutanesulfonyl groups, respectively. The terms triflate,tosylate, mesylate, and nonaflate are art-recognized and refer totrifluoromethanesulfonate ester, p-toluenesulfonate ester,methanesulfonate ester, and nonafluorobutanesulfonate ester functionalgroups and molecules that contain said groups, respectively.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl,phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations. The abbreviationscontained in said list, and all abbreviations utilized by organicchemists of ordinary skill in the art are hereby incorporated byreference.

The term “sulfate” is art recognized and includes a moiety that can berepresented by the general formula:

in which R₄₁ is as defined above.

The term “sulfonylamino” is art recognized and includes a moiety thatcan be represented by the general formula:

The term “sulfamoyl” is art-recognized and includes a moiety that can berepresented by the general formula:

The term “sulfonyl”, as used herein, refers to a moiety that can berepresented by the general formula:

in which R₄₄ is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl.

The term “sulfoxido” as used herein, refers to a moiety that can berepresented by the general formula:

in which R₄₄ is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.

A “selenoalkyl” refers to an alkyl group having a substituted selenogroup attached thereto. Exemplary “selenoethers” which may besubstituted on the alkyl are selected from one of —Se-alkyl,—Se-alkenyl, —Se-alkynyl, and —Se—(CH₂)_(m)—R₇, m and R₇ being definedabove.

Analogous substitutions can be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

As used herein, the definition of each expression, e.g. alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

The phrase “protecting group” as used herein means temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G.M. Protective Groups in Organic Synthesis, 2^(nd) ed.; Wiley: New York,1991).

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986–87, inside cover.

Compounds of the Invention

In certain embodiments, the present invention relates to a compoundrepresented by A:

wherein

X represents O, S, N(R), or C(R)₂;

R represents independently for each occurrence H, alkyl, aryl,heteroaryl, aralkyl, heteroaralkyl, formyl, acyl, alkoxycarbonyl,aralkoxycarbonyl, alkylaminocarbonyl, or aralkylaminocarbonyl;

R′ represents H, alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; orXR′ represents halide;

the stereochemical configuration at any stereocenter of a compoundrepresented by A may be R, S, or a mixture of these configurations; and

the enantiomeric excess of a compound represented by A is greater thanor equal to about 85%.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein X represents O or N(R).

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein R represents independently for each occurrence H, alkyl,aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, aralkylaminocarbonyl,or aralkylaminocarbonyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein R represents independently for each occurrence H.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein R′ represents H, alkyl, or aralkyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein R′ represents H.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein R represents independently for each occurrence H; and R′represents H.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein X represents O or N(R); and R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein X represents O or N(R); and R represents independently for eachoccurrence H.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein X represents O or N(R); and R′ represents H, alkyl, or aralkyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein X represents O or N(R); and R′ represents H.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H, alkyl,or aralkyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H; and R′ represents H, alkyl, or aralkyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H.

In certain embodiments, the compounds of the present invention arerepresented by general structure A and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H; and R′ represents H.

In certain embodiments, the present invention relates to a compoundrepresented by B:

wherein

X represents O, S, N(R), or C(R)₂;

R represents independently for each occurrence H, alkyl, aryl,heteroaryl, aralkyl, heteroaralkyl, formyl, acyl, alkoxycarbonyl,aralkoxycarbonyl, alkylaminocarbonyl, or aralkylaminocarbonyl;

R′ represents H, alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl,formyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl; or XR′ represents halide;

the stereochemical configuration at any stereocenter of a compoundrepresented by B may be R, S, or a mixture of these configurations; and

the enantiomeric excess of a compound represented by B is greater thanor equal to about 85%.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein X represents O or N(R).

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein R represents independently for each occurrence H, alkyl,aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, aralkylaminocarbonyl,or aralkylaminocarbonyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein R represents independently for each occurrence H.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein R′ represents H, aralkyl, formyl, acyl, alkoxycarbonyl,aralkoxycarbonyl, alkylaminocarbonyl, or aralkylaminocarbonyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein R′ represents H.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein R represents independently for each occurrence H; and R′represents H.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein X represents O or N(R); and R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein X represents O or N(R); and R represents independently for eachoccurrence H.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein X represents O or N(R); and R′ represents H, aralkyl, formyl,acyl, alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein X represents O or N(R); and R′ represents H.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H,aralkyl, formyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H; and R′ represents H, aralkyl, formyl, acyl,alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H.

In certain embodiments, the compounds of the present invention arerepresented by general structure B and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H; and R′ represents H.

In certain embodiments, the present invention relates to a compoundrepresented by C:

wherein

X represents O, S, N(R), or C(R)₂;

R represents independently for each occurrence H, alkyl, aryl,heteroaryl, aralkyl, heteroaralkyl, formyl, acyl, alkoxycarbonyl,aralkoxycarbonyl, alkylaminocarbonyl, or aralkylaminocarbonyl;

R′ represents H, alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; orXR′ represents halide;

the stereochemical configuration at any stereocenter of a compoundrepresented by C may be R, S, or a mixture of these configurations; and

the enantiomeric excess of a compound represented by C is greater thanor equal to about 85%.

In certain embodiments, the compounds of the present invention arerepresented by general structure C and the attendant definitions,wherein X represents O or N(R).

In certain embodiments, the compounds of the present invention arerepresented by general structure C and the attendant definitions,wherein R represents independently for each occurrence H, alkyl,aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, aralkylaminocarbonyl,or aralkylaminocarbonyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure C and the attendant definitions,wherein R represents independently for each occurrence H.

In certain embodiments, the compounds of the present invention arerepresented by general structure C and the attendant definitions,wherein R′ represents H, alkyl, or aralkyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure C and the attendant definitions,wherein R′ represents H.

In certain embodiments, the compounds of the present invention arerepresented by general structure C and the attendant definitions,wherein R represents independently for each occurrence H; and R′represents H.

In certain embodiments, the compounds of the present invention arerepresented by general structure C and the attendant definitions,wherein X represents O or N(R); and R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure C and the attendant definitions,wherein X represents O or N(R); and R represents independently for eachoccurrence H.

In certain embodiments, the compounds of the present invention arerepresented by general structure C and the attendant definitions,wherein X represents O or N(R); and R′ represents H, alkyl, or aralkyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure C and the attendant definitions,wherein X represents O or N(R); and R′ represents H.

In certain embodiments, the compounds of the present invention arerepresented by general structure C and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H, alkyl,or aralkyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure C and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H; and R′ represents H, alkyl, or aralkyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure C and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H.

In certain embodiments, the compounds of the present invention arerepresented by general structure C and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H; and R′ represents H.

In certain embodiments, the present invention relates to a compoundrepresented by

wherein

X represents O, S, N(R), or C(R)₂;

R represents independently for each occurrence H, alkyl, aryl,heteroaryl, aralkyl, heteroaralkyl, formyl, acyl, alkoxycarbonyl,aralkoxycarbonyl, alkylaminocarbonyl, or aralkylaminocarbonyl;

R′ represents H, alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl,formyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl; or XR′ represents halide;

the stereochemical configuration at any stereocenter of a compoundrepresented by D may be R, S, or a mixture of these configurations; and

the enantiomeric excess of a compound represented by D is greater thanor equal to about 85%.

In certain embodiments, the compounds of the present invention arerepresented by general structure D and the attendant definitions,wherein X represents O or N(R).

In certain embodiments, the compounds of the present invention arerepresented by general structure D and the attendant definitions,wherein R represents independently for each occurrence H, alkyl,aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, aralkylaminocarbonyl,or aralkylaminocarbonyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure D and the attendant definitions,wherein R′ represents independently for each occurrence H.

In certain embodiments, the compounds of the present invention arerepresented by general structure D and the attendant definitions,wherein R′ represents H, aralkyl, formyl, acyl, alkoxycarbonyl,aralkoxycarbonyl, alkylaminocarbonyl, or aralkylaminocarbonyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure D and the attendant definitions,wherein R′ represents H.

In certain embodiments, the compounds of the present invention arerepresented by general structure D and the attendant definitions,wherein R represents independently for each occurrence H; and R′represents H.

In certain embodiments, the compounds of the present invention arerepresented by general structure D and the attendant definitions,wherein X represents O or N(R); and R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure D and the attendant definitions,wherein X represents O or N(R); and R represents independently for eachoccurrence H.

In certain embodiments, the compounds of the present invention arerepresented by general structure D and the attendant definitions,wherein X represents O or N(R); and R′ represents H, aralkyl, formyl,acyl, alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure D and the attendant definitions,wherein X represents O or N(R); and R′ represents H.

In certain embodiments, the compounds of the present invention arerepresented by general structure D and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H,aralkyl, formyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure D and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H; and R′ represents H, aralkyl, formyl, acyl,alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure D and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H.

In certain embodiments, the compounds of the present invention arerepresented by general structure D and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H; and R′ represents H.

In certain embodiments, the present invention relates to a compoundrepresented by E:

wherein

X represents O, S, N(R), or C(R)₂;

R represents independently for each occurrence H, alkyl, aryl,heteroaryl, aralkyl, heteroaralkyl, formyl, acyl, alkoxycarbonyl,aralkoxycarbonyl, alkylaminocarbonyl, or aralkylaminocarbonyl;

R′ represents H, alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; orXR′ represents halide;

the stereochemical configuration at any stereocenter of a compoundrepresented by E may be R, S, or a mixture of these configurations; and

the enantiomeric excess of a compound represented by E is greater thanor equal to about 85%.

In certain embodiments, the compounds of the present invention arerepresented by general structure E and the attendant definitions,wherein X represents O or N(R).

In certain embodiments, the compounds of the present invention arerepresented by general structure E and the attendant definitions,wherein R represents independently for each occurrence H, alkyl,aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, aralkylaminocarbonyl,or aralkylaminocarbonyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure E and the attendant definitions,wherein R represents independently for each occurrence H.

In certain embodiments, the compounds of the present invention arerepresented by general structure E and the attendant definitions,wherein R′ represents H, alkyl, or aralkyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure E and the attendant definitions,wherein R′ represents H.

In certain embodiments, the compounds of the present invention arerepresented by general structure E and the attendant definitions,wherein R represents independently for each occurrence H; and R′represents H.

In certain embodiments, the compounds of the present invention arerepresented by general structure E and the attendant definitions,wherein X represents O or N(R); and R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure E and the attendant definitions,wherein X represents O or N(R); and R represents independently for eachoccurrence H.

In certain embodiments, the compounds of the present invention arerepresented by general structure E and the attendant definitions,wherein X represents O or N(R); and R′ represents H, alkyl, or aralkyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure E and the attendant definitions,wherein X represents O or N(R); and R′ represents H.

In certain embodiments, the compounds of the present invention arerepresented by general structure E and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H, alkyl,or aralkyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure E and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H; and R′ represents H, alkyl, or aralkyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure E and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H.

In certain embodiments, the compounds of the present invention arerepresented by general structure E and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H; and R′ represents H.

In certain embodiments, the present invention relates to a compoundrepresented by F:

wherein

X represents O, S, N(R), or C(R)₂;

R represents independently for each occurrence H, alkyl, aryl,heteroaryl, aralkyl, heteroaralkyl, formyl, acyl, alkoxycarbonyl,aralkoxycarbonyl, alkylaminocarbonyl, or aralkylaminocarbonyl;

R′ represents H, alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl,formyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl; or XR′ represents halide;

the stereochemical configuration at any stereocenter of a compoundrepresented by F may be R, S, or a mixture of these configurations; and

the enantiomeric excess of a compound represented by F is greater thanor equal to about 85%.

In certain embodiments, the compounds of the present invention arerepresented by general structure F and the attendant definitions,wherein X represents O or N(R).

In certain embodiments, the compounds of the present invention arerepresented by general structure F and the attendant definitions,wherein R represents independently for each occurrence H, alkyl,aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, aralkylaminocarbonyl,or aralkylaminocarbonyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure F and the attendant definitions,wherein R represents independently for each occurrence H.

In certain embodiments, the compounds of the present invention arerepresented by general structure F and the attendant definitions,wherein R′ represents H, aralkyl, formyl, acyl, alkoxycarbonyl,aralkoxycarbonyl, alkylaminocarbonyl, or aralkylaminocarbonyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure F and the attendant definitions,wherein R′ represents H.

In certain embodiments, the compounds of the present invention arerepresented by general structure F and the attendant definitions,wherein R represents independently for each occurrence H; and R′represents H.

In certain embodiments, the compounds of the present invention arerepresented by general structure F and the attendant definitions,wherein X represents O or N(R); and R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure F and the attendant definitions,wherein X represents O or N(R); and R represents independently for eachoccurrence H.

In certain embodiments, the compounds of the present invention arerepresented by general structure F and the attendant definitions,wherein X represents O or N(R); and R′ represents H, aralkyl, formyl,acyl, alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure F and the attendant definitions,wherein X represents O or N(R); and R′ represents H.

In certain embodiments, the compounds of the present invention arerepresented by general structure F and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H,aralkyl, formyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure F and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H; and R′ represents H, aralkyl, formyl, acyl,alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl.

In certain embodiments, the compounds of the present invention arerepresented by general structure F and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H.

In certain embodiments, the compounds of the present invention arerepresented by general structure F and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H; and R′ represents H.

In certain embodiments, the present invention relates to a compoundrepresented by any of the structures outlined above, wherein theenantiomeric excess of said compound is greater than or equal to about90%.

In certain embodiments, the present invention relates to a compoundrepresented by any of the structures outlined above, wherein theenantiomeric excess of said compound is greater than or equal to about95%.

In certain embodiments, the present invention relates to a compoundrepresented by any of the structures outlined above, wherein saidcompound is a single stereoisomer.

In certain embodiments, the present invention relates to a compoundrepresented by any of the structures outlined above, wherein saidcompound is in the form of a salt.

In certain embodiments, the present invention relates to a formulation,comprising a compound represented by any of the structures outlinedabove; and a pharmaceutically acceptable excipient.

In certain embodiments, the present invention relates to an oligopeptideor a polypeptide, comprising a compound represented by any of thestructures outlined above.

Methods of the Invention

In certain embodiments, the present invention relates to a method ofresolving into individual enantiomers a mixture of diastereomers of acompound represented by structure A, B, C, D, E, or F, comprising thesteps of:

(a) using chromatography to obtain an individual pair of enantiomers ofa compound represented by structure A, B, C, D, E, or F from a mixtureof diastereomers of said compound; and

(b) using enzymatic hydrolysis to obtain a single enantiomer of saidcompound from the individual pair of enantiomers of said compound.

In certain embodiments, the present invention relates to theaforementioned resolution method, wherein (R)₂N represents(alkoxycarbonyl)HN in the mixture of diastereomers.

In certain embodiments, the present invention relates to theaforementioned resolution method, wherein (R)₂N represents(tert-butyloxycarbonyl)HN in the mixture of diastereomers.

In certain embodiments, the present invention relates to theaforementioned resolution method, wherein (R)₂N represents (acyl)HN inthe the individual pair of enantiomers subjected to enzymatichydrolysis.

In certain embodiments, the present invention relates to theaforementioned resolution method, wherein (R)₂N represents (acetyl)HN inthe the individual pair of enantiomers subjected to enzymatichydrolysis.

In certain embodiments, the present invention relates to theaforementioned resolution method, wherein the enzyme used is porcinekidney acylase I.

In certain embodiments, the present invention relates to theaforementioned resolution method, wherein (R)₂N represents(alkoxycarbonyl)HN in the mixture of diastereomers; and (R)₂N represents(acyl)HN in the the individual pair of enantiomers subjected toenzymatic hydrolysis.

In certain embodiments, the present invention relates to theaforementioned resolution method, wherein (R)₂N represents(tert-butyloxycarbonyl)HN in the mixture of diastereomers; and (R)₂Nrepresents (acetyl)HN in the the individual pair of enantiomerssubjected to enzymatic hydrolysis.

In certain embodiments, the present invention relates to theaforementioned resolution method, wherein (R)₂N represents(alkoxycarbonyl)HN in the mixture of diastereomers; (R)₂N represents(acyl)HN in the the individual pair of enantiomers subjected toenzymatic hydrolysis; and the enzyme used is porcine kidney acylase I.

In certain embodiments, the present invention relates to theaforementioned resolution method, wherein (R)₂N represents(tert-butyloxycarbonyl)HN in the mixture of diastereomers; (R)₂Nrepresents (acetyl)HN in the the individual pair of enantiomerssubjected to enzymatic hydrolysis; and the enzyme used is porcine kidneyacylase I.

In certain embodiments, the present invention relates to a method ofsynthesizing a non-native oligopeptide, polypeptide or protein withenhanced hydrophobicity relative to a native oligopeptide, polypeptideor protein, comprising the step of using a compound represented bystructure A, B, C, D, E, or F in place of a leucine or valine in asynthesis of an oligopeptide, polypeptide or protein.

In certain embodiments, the present invention relates to theaforementioned method of synthesizing a non-native oligopeptide,polypeptide or protein with enhanced hydrophobicity, wherein thesynthesis is automated.

In certain embodiments, the present invention relates to a method ofenhancing the hydrophobicity of an oligopeptide, polypeptide or protein,comprising the step of replacing a leucine or valine in an oligopeptide,polypeptide or protein with a compound represented by structure A, B, C,D, E, or F.

In certain embodiments, the present invention relates to a method ofsynthesizing a trifluoromethyl-containing analogue of norvaline orvaline, comprising the steps of:

(a) oxidizing a protected serine or homoserine to give an aldehyde;

(b) reacting the aldehyde with trimethyl(trifluoromethyl)silane andfluoride to give a secondary alcohol;

(c) acylating the secondary alcohol using an aryl chlorothionoformate togive a thionocarbonate; and

(d) reducing the thionocarbonate using a tin hydride and an initiator togive a trifluoromethyl-containing analogue of norvaline or valine.

In certain embodiments, the present invention relates to a method ofsynthesizing a trifluoromethyl-containing analogue of leucine,comprising the steps of:

(a) oxidizing a protected homoserine to give an aldehyde;

(b) reacting the aldehyde with trimethyl(trifluoromethyl)silane andfluoride to give a secondary alcohol;

(c) oxidizing the secondary alcohol to give a trifluoromethyl ketone;

(d) reacting the trifluoromethyl ketone with(methylene)triphenylphosphine to give an alkene; and

(e) hydrogenating the alkene to give a trifluoromethyl-containinganalogue of leucine.

In certain embodiments, the present invention relates to a method ofsynthesizing protected 5,5,5,5′,5′,5′-hexafluoroleucine, comprising thesteps of:

(a) reacting an oxazolidine aldehyde derived from serine with ahexafluoroisopropylidene ylide to give an oxazolidine1,1-bis(trifluoromethyl)alkene;

(b) hydrogenating the oxazolidine 1,1-bis(trifluoromethyl)alkene to givean oxazolidine 1,1-bis(trifluoromethyl)alkane;

(c) hydrolyzing the oxazolidine 1,1-bis(trifluoromethyl)alkane to give aprotected amino alcohol; and

(d) oxidizing the protected amino alcohol to give protected5,5,5,5′,5′,5′-hexafluoroleucine.

In certain embodiments, the present invention relates to theaforemetioned method of synthesizing protected5,5,5,5′,5′,5′-hexafluoroleucine, wherein the reagents for step (a)comprise triphenylphosphine and [(CF₃)₂C]₂S₂; the reagents for step (b)comprise hydrogen and 10% palladium on carbon; the reagents for step (c)comprise toluenesulfonic acid and methanol; and the reagents for step(d) comprise pyridinium dichromate.

In certain embodiments, the present invention relates to a method ofpreparing a compound represented by 6, comprising the steps depicted inScheme 1:

Exemplification

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

EXAMPLE 1

General Experimental Procedures

Melting points were determined in open capillaries on a MEL-TEMP IIapparatus (Laboratory Devices, Inc., Holliston, Mass.) and areuncorrected. All reactions requiring non-aqueous conditions wereperformed in oven-dried glassware under positive pressure of argon.Flash column chromatography was performed by forced flow of solventusing Kieselgel 60 SiO₂ (230–240 mesh) gel (EM Science) packed intoglass columns using standard litertaure procedures. Still, W. C.; Kahn,M.; Mitra, A. J. Org. Chem. 1978, 43, 2923. Analytical thin layerchromatography was performed using E. Merck silica gel Kieselgel 60 F₂₅₄(0.25 mm) plates. Compounds were visualized by UV light, exposure toiodine vapour or by staining with a ninhydrin solution followed byheating. Reagents and solvents were of reagent grade or better and wereobtained from Aldrich Chemical Co., Fluka Chemie AG, Lancaster Synthesisor Novabiochem Corp. Deuterated solvents were obtained from CambridgeIsotope Laboratories.

Infra-red spectra were obtained on a Mattson 1000 FT-IR instrument witha 4 cm⁻1 bandpass. Spectra of solid samples were obtained as solidthin-films or dissolved in thin layers of organic solvents between NaClplates. Mass Spectra were obtained on a Hewlett Packard GC-MS (Model5988A) with a dip-probe using conditions as indicated. Nuclear magneticresonance spectra were recorded on a Bruker AM-300 or a Bruker DPX-300instrument in standard deuterated solvents. Optical rotations weremeasured using an AUTOPOL IV digital polarimeter (Rudolph ResearchAnalytical, N.J.).

EXAMPLE 2

Synthesis of Bis-trifluoromethyl Olefin (2)

Typical procedure for the coupling reaction: To a stirred solution ofthe Garner aldehyde 1 (7.0 g, 31.0 mmol) and PPh₃ (57 g, 217 mmol) indry Et₂O (300 mL) was added2,2,4,4-tetrakis-(trifluoromethyl)-1,3-dithietane (39.5 g, 108.5 mmol)at −78° C. under argon. The mixture was stirred for 3 d while beingslowly warmed to room temperature. The reaction slowly accumulated aninsoluble white solid which was filtered and the filtrate concentrated.The residue was further dissolved in n-pentane (300 mL) and filteredagain to remove insoluble impurities. After removal of the solvent, theresidue was subjected to flash column chromatography usingn-pentane/Et₂O (6/1) as eluant to give pure 2 as a pale yellow oil (10.4g, 92%). ¹H NMR (300 MHz, CDCl₃) δ 6.70 (d, 1H, J=8.7 Hz), 4.81 (bs,1H), 4.23 (dd, 1H, J=6.9 Hz, 9.3 Hz), 3.79 (dd, 1H, J=3.9 Hz, 9.3 Hz),1.65 (s, 3H), 1.56 (s, 3H), 1.42 (s, 9H); ¹⁹F NMR (282.6 MHz,CDCl₃/CFCl₃) δ −65.01 (d, 3F, J=5.9 Hz), −58.44 (d, 3F, J=5.9 Hz); FT-IR(film, V_(max), cm⁻¹) 2983m, 2935m, 2885w, 1713s, 1479W, 1460w, 1379s,1230s, 1165s, 1110m, 971m; [α]_(D) ^(26.1)=+12.3° (c 1.7, CHCl₃); GC-MS(CI, CH₄): 364 (1, [M+1]⁺), 336 (18), 308 (100), 288 (98), 264 (37), 102(2), 57 (9).

EXAMPLE 3

Synthesis of Oxazolidine (3)

A 500 mL round bottomed flask was charged with a solution of 2 (10.3 g,28.3 mmol) in THF (250 mL) and 10% Pd/C (40 g). The reaction flask waspurged with argon and hydrogen sequentially and stirred under hydrogenat room temperature until uptake of H₂ ceased (24 hours). The catalystwas then separated from the reaction mixture by filtration (and can beused again). The filtrate was dried over anhydrous MgSO₄ andconcentrated by rotary evaporation to give 3 (10.1 g, 98% yield) as apale yellow oil. ¹H NMR (300 MHz, CDCl₃) δ 4.23 (4.05) (m, 1H), 4.00(dd, 1H, J=5.4 Hz, 9.3 Hz), 3.73 (d, 1H, J=9.3 Hz), 3.58 (3.05) (m, 1H),2.18 (2.01) (m, 2H), 1.62 (1.58) (s, 3H), 1.48 (br. s, 12H); ¹³C NMR(75.5 MHz, CDCl₃) δ 153.22 (151.51) (C═O), 123.89 (q, 2×CF₃,¹J_(CF)=284.0), 94.47 (94.03) (C), 80.85 (80.73) (C), 67.26 (66.65)(CH₂), 55.58 (55.12) (CH), 45.44 (45.12) (quintet, CH, ²J_(CF)=27.2 Hz),28.98 (28.00) (CH₂), 28.25 (3×CH₃), 27,58 (26.90) (CH₃), 24.15 (22.86)(CH₃); ¹⁹F NMR (282.6 MHz, CDCl₃/CFCl₃) δ−67.68–−68.42 (m); FT-IR (film,v_(max), cm⁻¹): 2984m, 2941m, 2884w, 1704s, 1457m, 1393s, 1258s, 1168s,1104s, 847m; [α]_(D) ^(22.4)=+17.5° (c 0.4, CHCl₃); GC-MS (CI, CH₄): 366(4, [M+1]⁺), 338 (16), 310 (100), 290 (48), 266 (48), 57 (8).

EXAMPLE 4

Synthesis of N-Boc-5,5,5,5′,5′,5′-(S)-Hexafluoroleucinol (4)

To a solution of3 (10.1 g, 27.6 mmol) in CH₂Cl₂ (30 mL) was added 10 mLof trifluoroacetic acid (TFA). The reaction mixture was stirred at roomtemperature for 5 min. After removal of the solvent and TFA, the residuewas partitioned between 150 mL of ethyl ether and 100 mL of H₂O. Theorganic layer was washed with water (20 mL×4), dried over MgSO₄, andconcentrated to give 4 (7.2 g, 80% yield) as a white solid. The aqueouslayers contain a completely deprotected product due to cleavage of theBOC moiety as evidenced by ninhydrin active material. Thishexafluoroamino alcohol can be converted back to 4 by protecting thefree amine group as a BOC amide. ¹H NMR (300 MHz, CDCl₃) δ 5.03 (d, 1H,J=8.1 Hz), 3.84 (m, 1H), 3.70 (m, 2H), 3.20 (m, 1H), 3.10 (br.s, 1H),1.98 (m, 2H), 1.45 (s, 9H); ¹³C NMR (75.5 MHz, CDCl₃) δ 156.57 (C═O),124.00 (q, 2×CF₃, ¹J_(CF)=284.0 Hz), 80.58 (C), 66.08 (CH₂), 50.57 (CH),45.09 (m, CH, ²J_(CF)=28.1 Hz), 28.38 (3×CH₃), 26.44 (CH₂); ¹⁹F NMR(282.6 MHz, CDCl₃/CFCl₃) δ −67.96 (m), −68.46 (m); FT-IR (KBr pellet,v_(max), cm⁻¹) 3397s (br), 3253s, 3068m, 2981s, 2948m, 1686s, 1552s,1369s, 1289s, 1174s, 1145s, 1055s; [α]_(D) ^(22.9)=−14.4° (c 1.0,CH₃OH); GC-MS (CI, CH₄); 326 (8, [M+1]⁺), 298 (14), 270 (100), 226 (20),57 (2); m.p.=114–115° C.

EXAMPLE 5

Synthesis of N-Boc-5,5,5,5′,5′,5′-(S)-Hexafluoroleucine (5)

A mixture of 4 (7.1 g, 21.8 mmol) and pyridinium dichromate (33 g, 88mmol) in DMF (150 mL) was stirred under argon at room temperature for 24hrs. before 150 mL of H₂O was added. The mixture was then extracted withethyl ether (400 mL×2). The combined ether layers were washed with 1 NHCl (80 mL×2) and concentrated until about 150 mL of solution left. Thissolution was washed with 5% NaHCO₃ (150 mL×3). The combined aqueouslayers were acidified to pH 2 with 3 N HCl, extracted with ether again(400 mL×2). The ether layers were then dried over MgSO₄ and concentratedto give 5 (5.2 g, 70%) as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 7.36(5.21) (d, 1H, J=6.3 Hz), 4.41 (m, 1H), 3.37 (m, 1H), 2.43–2.11 (br. m,2H), 1.47 (s, 9H); ¹⁹F NMR (282.6 MHz, CDCl₃/CFCl₃) δ −67.87–−68.23 (m);FT-IR (KBr pellet, v_(max), cm⁻¹) 3358–2500m (br.), 3245s, 3107m, 2989s,2980m, 1725s, 1712s, 1657s, 1477s, 1458s, 1404s, 1296s, 1277s, 1258s,916m; [α]_(D) ^(21.8)=−23.0° (c 1.0, CH₃OH); GC-MS (CI, CH₄): 340 (21,[M+1]⁺), 312 (7), 284 (100), 264 (16), 240 (19), 57 (39); m.p.=85–91° C.

EXAMPLE 6

Synthesis of 5,5,5,5′,5′,5′-(S)-Hexafluoroleucine (6)

A solution of 5 (581 mg, 1.7 mmol) in 5 mL of TFA/CH₂Cl₂ (2/3) wasstirred for 30 min. After removal of the solvents, the residue waspartitioned between 1 N HCl (10 mL×3) and ethyl ether (10 mL). Thecombined aqueous layers were freeze dried to give 6 (446 mg, 95% yield)as a white solid.

EXAMPLE 7

Synthesis of Dipeptide (8)

To a stirred solution of 5 (11 mg, 0.03 mmol) in anhydrous DMF (1 mL)was added diisopropyl ethyl amine (13 mg, 0.1 mmol), HBTU (13 mg, 0.03mmol), and H-Ser(t-Bu)-OMe HCl (14 mg, 0.065 mmol) sequentially. Themixture was stirred at room temperature for 40 min before 6 mL of H₂Owas added. The reaction mixture was extracted with ether (15 ml) and theorganic layer was futher washed with 1 N HCl (5 mL×2) and 5% NaHCO₃solution (5 ml), dried over MgSO₄, and concentrated to afford 8 (13 mg,87% yield) as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 6.68 (d, 1H,J=8.1 Hz), 5.21 (d, 1H, J=8.1 Hz), 4.64 (m, 1H), 4.40 (m, 1H), 3.86 (dd,1H, J=2.7 Hz, 9.3 Hz), 3.76 (s, 3H), 3.56 (dd, 1H, J=3.3 Hz, 9.3 Hz),3.50 (m, 1H), 2.33–2.10 (br. m, 2H), 1.45 (s, 9H), 1.14 (s, 9H).

EXAMPLE 8

Incorporation of 5,5,5,5′,5′,5′,5′-hexafluoroleucine into Peptides

The incorporation of hexafluoroleucine in a 30-residue peptide with thesequence given below has been achieved. Leucines in bold are5,5,5,5′,5′,5′-(S)-hexafluoroleucine.

Peptide 1: Ac-NH-AQLKKELQALKKENAQLKWELQALKKELAQ-CONH₂

The MALDI-MS of purified peptide 1 (Calc. 4316.8, found 4317.1) confirmsthe purity and identity of the peptide. Circular dichroism dataindicates that the peptide can readily adopt an alpha helical secondarystructure (characterisitic minima at 208 and 222 nm). Furtherbiophysical studies with these peptides are in progress.

Peptide Synthesis

Peptides were prepared using the N-tert-butyloxycarbonyl (t-Boc) aminoacid derivatives for Merrifield manual solid-phase synthesis (MBHAresin) using the in-situ neutralization/HBTU protocol on a 0.2 mmolscale. Schnolzer, M.; Alewood, P.; Jones, A.; Alewood.; D, Kent, S. B.Int. J. Pept. Protein Res. 1992, 40, 180–193. N-α-Boc-α-S-amino acidswere used with the following side chain protecting groups: Arg(Tos),Asp(OBzl), Asn(Xan), Gln(Xan), Glu(OBzl) and Lys(2-Cl-Z). Peptidecoupling reactions were carried out with 4-fold excess (0.8 mmol) ofactivated amino acid for at least 15 min. Peptides were cleaved from theresin using high HF conditions (90% anhydrous HF/10% anisole at 0° C.for 1.5 hours) with simultaneous removal of the side chain protectinggroups. Tam J. P.; Merrifield, R. B. In The Peptides; Udenfriend, S.,Meienhofer, J. Eds.; Academic Press Inc.: New York, 1987; Vol. 9, p 185.

In the case of hexafluoroleucine, the coupling time was extended to 2hrs. The extent of reaction was verified by a Kaiser test after eachcoupling. The N-terminal was acetylated by treatment with 1:4 aceticanhydride/DMF and 6 eq. of diisopropylethylamine. The formyl protectinggroup on the tryptophan residue was removed by treating the resin with1:10 piperidine in DMF solution. Peptides were cleaved from the resin byusing high HF conditions (90% anhydrous HF/10% anisole at 0° C. for 1.5h). Crude peptides were extracted with 25% acetic acid and lyophilized.Freeze dried material was dissolved in 0.1% TFA, desalted and purifiedby reversed phase HPLC [Vydac C4 column with a 30 min linear gradient ofacetonitrile/H₂O/0.1% TFA at 8.0 mL/min].

HH: An aqueous solution of H (10 mg, 2.64 μmol) in 50 mM Tris (pH 8.50)and 6 M Gdn HCl (total volume: 0.75 mL) was stirred overnight at roomtemperature. The reaction was quenched by addition of 250 μL glacialacetic acid and diluted with 1 mL water. The mixture was directlypurified by reversed phase HPLC. The fractions containing HH werecollected and lyophilized to deliver 9.0 mg (90%) of HH. MALDI-MS:MW_(calcd)=7556.8, found: 7561.

FF: An aqueous solution of F (14 mg, 3.09 μmol) in 50 mM Tris (pH 8.50)and 6.5 M Gdn HCl (total volume: 1 mL) was stirred overnight at roomtemperature. The reaction was quenched by addition of 300 μL glacialacetic acid and diluted with 1.5 mL water. The mixture was directlypurified by reversed phase HPLC. The fractions containing FF were pooledand lyophilized to deliver 12.1 mg (86%) of FF. MALDI-MS:MW_(calcd)=9066, found: 9076.3.

HF: To an aqueous solution of H (8 mg, 2.11 μmol) in MOPS buffer (pH7.50) was added 5,5′-dithiobis(2-nitrobenzoic acid) (20 mg, 50.4 μmol).The reaction was stirred for 15 minutes and then quenched by theaddition of 300 μL of neat TFA. The reaction mixture was then extractedwith Et₂O (4×10 mL). The aqueous layer was then directly injected into areversed-phase C18 column and purified. The fractions containing themixed disulfide of the Ellman's reagent and H were combined andlyophilized to obtain 8.4 mg of the desired product (95%). The mixeddisulfide (8 mg, 1.92 μmol) was dissolved in a pH 1.50 solutioncontaining F (17.4 mg, 3.84 μmol). The pH was carefully adjusted to 5.10by sequential addition of 0.1 N NaOH solution. The reaction was allowedto proceed for 20 minutes and then quenched by addition of 300 μL TFA.The reaction mixture was then directly purified by reversed phase HPLCto obtain 10 mg of pure HF (62.6%). Nearly 25% of the starting mixeddisulfide was recovered unreacted. MALDI-MS: MW_(calcd): 8310.4, found:8317.

Purification

Peptides were desalted and purified by reversed phase HPLC [Vydac C₄column using a 30 min linear gradient of 34–47% acetonitrile/H₂O/0.1%TFA at 8.0 mL/min]. Peptide 1 eluted at ˜43.2% acetonitrile/H₂O/0.1% TFA(˜30.0 min. elution time).

EXAMPLE 9

Circular Dichroism

Circular dichroism spectra were obtained on a JASCO J-715spectropolarimeter fitted with a PTC-423S single position Peltiertemperature controller. Buffer conditions were usually 10 mM phosphate(pH 7.40), 137 mM NaCl, 2.7 mM KCl unless otherwise noted. Thespectrometer was calibrated with an aqueous solution of recrystallizedd₁₀-(+)-camphorsulfonic acid at 290.5 nm. The concentrations of thepeptide stock solutions were determined by amino-acid analysis or bymeasuring tryptophan absorbance in 6 M Gdn HCl (assuming an extinctioncoefficient of 5600 M⁻¹ cm⁻¹ at 281 nm). Edelhoch, H. Biochemistry 1967,6, 1948. Mean residue ellipticities (deg cm² dmol⁻¹) were calculatedusing the relation:[θ]=θ_(obs)×MRW/10lc  (1)wherein θ_(obs); is the measured signal (ellipticity) in millidegrees, lis the optical pathlength of the cell in cm, c is concentration of thepeptide in mg/mL and MRW is the mean residue molecular weight (molecularweight of the peptide divided by the number of residues).

Thermal denaturation studies were carried out at the concentrationsindicated by monitoring the change in [θ]₂₂₂ as a function oftemperature. Temperature was increased in steps of 0.5° C. with anintervening equilibration time of 120s. Data was collected over 16 s perpoint. The T_(m) was determined from the minima of the first derivativeof [θ]₂₂₂ with T⁻¹, where Tis in K.

EXAMPLE 10

Analytical Ultracentrifugation

Apparent molecular masses were determined by sedimentation equilibriumon a Beckman XL-A ultracentrifuge. Loading peptide concentrations were2–15 μM in 10 mM phosphate (pH 7.40), 137 mM NaCl, 2.7 mM KCl. Thesamples were centrifuged at 32 000 and 26 000 rpm for 18 hours at 10° C.before absorbance scans were performed.

Data obtained at 10° C. were fit globally to the following equation (2)that describes the sedimentation of a homogeneous species:Abs=A′ exp(H×M[x ² −x ₀ ²])+B  (2)wherein Abs=absorbance at radius x, A′=absorbance at reference radiusx₀, H=(1−{overscore (V)}ρ)ω²/2RT, {overscore (V)}=partial specificvolume=0.758 mL/g, ρ=density of solvent=1.0017 g/mL, ω=angular velocityin radians/sec, and M=apparent molecular weight, B=solvent absorbance(blank). We estimated partial specific volume using amino acidcomposition (Cohn, E. J., Edsall, J. T. Proteins, Amino Acids andPeptides as Ions and Dipolar Ions. New York, Reinhold, 1943)substituting leucine for hexafluoroleucine in the case of HF and FF forlack of available data.

EXAMPLE 11

Calculation of Free Energies

The thermodynamic cycle used for calculating ΔG_(spec) (free energy ofspecificity for the formation of homodimers) is depicted below. Thesuperscripts U and F refer to the unfolded and folded statesrespectively of the disulfide bonded dimeric peptides.

K_(redox) is the equilibrium constant for the redox reaction. K_(random)is the equilibrium constant for the chance pairing of FF, HH and HFpeptides and is assumed to be 2 as there are two equivalent ways for theformation of the heterodimer HF but only one way to form each homodimer.O'Shea, E. K.; Rutkowski, R.; Kim, P. S. Cell 1992, 68, 69–708. FFFF^(F)is the dimer of the disulfide bonded dimer FF and K_(tetramer) is theequilibrium constant for it's formation. K_(redox) was estimated fromequilibrium ratios of HH, FF and HF.

$K_{redox} = \frac{\left\lbrack {HF}^{F} \right\rbrack}{{\left\lbrack {FF}^{F} \right\rbrack^{0.5}\left\lbrack {HH}^{F} \right\rbrack}^{0.5}}$ΔG_(spec)(for homodimers)=−{ΔG_(redox)+RT In 2}

EXAMPLE 12

Calculation of Free Energy of Unfolding for Homomdimer

The free energy of unfolding for HH was determined by assuming a twostate equilibrium between folded and unfolded states:

where F_(HH) is the folded species and U_(HH) represents the fullyunfolded HH. Data were obtained by monitoring [θ]₂₂₂ as a function ofGdn HCl concentration. Data were analyzed by the linear extrapolationmethod to yield the free energy of unfolding. The equilibrium constantand therefore ΔG° are easily determined from the average fraction ofunfolding. Assuming that the linear dependence of ΔG° with denaturantconcentration in the transition region continues to zero concentration,the data can be extrapolated to obtain ΔG*_(H) ₂ _(O)°, the free energydifference in the absence of denaturant. Pace, C. N. Methods in Enzymol.1995, 259, 538–554; and Tanford, C. Adv. Protein Chem. 1962, 17, 69–165.

Sedimentation equilibrium experiments suggest FF is a tetramer (dimer ofthe disulfide bonded dimer) in the 2–15 μM concentration range.Therefore, we used a unfolded monomer-folded dimer equilibrium tocalculate ΔG° of unfolding:

where K_(d)=[UFF]²/[F_(FF)] (U_(FF)=unfolded FF and F_(FF)=folded dimerof FF with 4 helices). Since the total peptide concentration P_(t), canbe given by P_(t)=2 [F_(FF)]+[U_(FF)], the observed CD signal Y_(obs)can be described in terms of folded and unfolded baselines, Y_(fol) andY_(unfol), respectively by the following expression.

$\begin{matrix}{Y_{obs} = {{\left( {Y_{unfol} - Y_{fol}} \right)\frac{\sqrt{K_{d}^{2} + {8K_{d}P_{1}}} - K_{d}}{4P_{1}}} + Y_{fol}}} & (2)\end{matrix}$

Additionally, K_(d) can be expressed in terms of the free energy ofunfolding.K_(d)=exp(−ΔG°=/RT)  (3)

Assuming that the apparent free energy difference between folded F_(FF)and unfolded U_(FF) states is linearly dependent on the Gdn HClconcentration, ΔG° can be written as:ΔG°=ΔG*_(H) ₂ _(O) °−m[Gdn.HCl]  (4)

where ΔG*_(H) ₂ _(O)° is the free energy difference in the absence ofdenaturant and m is the dependency of the unfolding transition withrespect to the concentration of Gdn HCl. The data was fit for twoparameters, ΔG*_(H) ₂ _(O)°, and m by nonlinear least squares fitting.

EXAMPLE 13

N-Boc-4,4,4-trifluorovalinol (2)

To a suspension of Boc-DL-trifluorovaline (1.30 g, 4.79 mmol) and NaHCO₃(1.21 g, 14.37 mmol) in 20 mL of dry DMF was added 0.33 mL of CH₃I (5.27mmol) at room temperature under argon. The resulting mixture was stirredfor 5 h and then partitioned between 75 mL of ethyl acetate and 50 mL ofwater. The organic layer was washed with water (3×50 mL), dried overMgSO₄, and concentrated to yield 1.36 g (95%) of theBoc-DL-trifluorovaline methyl ester as a pale-yellow oil.

The Boc-TFV methyl ester (855 mg, 3 mmol) was dissolved in 20 mL ofmethanol, and NaBH4 (681 mg, 18 mmol) was added in small portions at 0°C. The reaction mixture was stirred overnight at room temperature andthen diluted with 80 mL of ethyl acetate, washed with water (3×50 mL),and dried over MgSO₄. After removal of the solvent, the crude product(Boc-trifluorovalinol) was chromatographed on a silica gel column(silica gel, 300 g) using n-pentane/Et₂O (1:1) as eluant to give 452 mgof 2a as a pale-yellow solid (58%) and 214 mg of 2b as a white solid(28%).

(2S,3S)-, (2R,3R)-N-Boc-4,4,4-trifluorovalinol (2a)

¹H NMR (300 MHz, CDCl₃) δ 5.04 (d, 1H, J=9.3 Hz), 4.02 (m, 1H), 3.62 (m,3H), 2.61 (m, 1H), 1.44 (s, 9H), 1.15 (d, 3H, J=7.2 Hz); ¹³C NMR (75.5MHz, CDCl₃) δ 156.20 (C═O), 127.83 (q, CF₃, ¹J_(CF)=279.9 Hz), 80.26(C), 62.78 (CH₂), 51.09 (CH), 38.47 )q, CH, ²J_(CF)=25.6 Hz), 28. 40(3×CH₃), 8.76 (CH₃); ¹⁹F NMR (282.6 MHz, CDCl₃/CFCl₃) δ −70.63 (d, 3F,J=9.0 Hz); FT-IR (KBr pellet, v_(max), cm⁻¹) 3435s, 3300s, 2990s, 2979m,2954m, 1691s, 1539s, 1537s, 1265s, 1172s, 1125; GC-MS (Cl, CH₄): 258(14, [M+1]⁺), 242 (4), 202 (100), 158 (37), 57 (14).

(2S,3R)-, (2R,3S)-N-Boc-4,4,4-trifluorovalinol (2b)

¹H NMR (300 MHz, CDCl₃) δ 5.11 (d, 1H, J=8.4 Hz), 3.80 (m, 1H), 3.66 (m,2H), 3.45 (t, 1H, J=5.7 Hz), 2.53 (m, 1H), 1.42 (s, 9H), 1.15 (d, 3H,J=7.2 Hz); ¹³C NMR (75.5 MHz, CDCl₃) δ 156.43 (C═O), 127.91 (q, CF₃,¹J_(CF)=280.2 Hz), 80.30 (C), 62.92 (CH₂), 52.56 (CH), 38.89 (q, CH,²J_(CF=)24.8 Hz), 28. 40 (3 ÅCH₃), 10.59 (CH₃); ¹⁹F NMR (282.6 MHz,CDCl₃/CFCl₃) δ −68.76 (d, 3F, J=8.5 Hz); FT-IR (film, v_(max) cm⁻¹):3436s, 3302s, 3012m, 2990m, 2954m, 1691s, 1532s, 1265s, 1172s, 1127s;GC-MS (Cl, CH₄): 258 (14, [M+1]⁺), 242 (4), 202 (100), 182 (8), 57 (14).

EXAMPLE 14

(2S,3S)-, (2R,3R)-N-Ac-4,4,4-trifluorovaline (3a)

A solution of alcohol 2a (257 mg, 1 mmol) in 4 mL of dry DMF was treatedwith PDC (2.26 g, 6 mmol) at room temperature under argon and stirredovernight. The reaction mixture was then diluted with 20 mL of diethylether/30 mL of saturated NaHCO₃ solution. The organic layer was washedwith 10 mL of saturated NaHCO₃. The combined aqueous layers wereacidified to pH 2 with 3 N HCl and extracted with diethyl ether (2×50mL). The combined organic layers were dried over MgSO₄ and concentratedto yield 176 mg of the corresponding Boc-trifluorovaline (65%).

Boc-TFV (176 mg, 0.65 mmol) was treated with 4 mL of 40% trifluoroaceticacid in CH₂Cl₂ for 10 min. After removal of the solvent, the residue wasdissolved in 2 mL of water, treated with NaOH (260 mg, 6.5 mmol) at 0°C., followed by dropwise addition of acetic anhydride (0.13 mL, 1.3mmol). The reaction mixture was stirred at 0° C. for 30 min before itwas allowed to warm to room temperature. After stirring for another 1.5h, the mixture was diluted with 10 mL of water, acidified to pH 2 with 1N HCl, and extracted with ethyl acetate (2×60 mL). The combined organiclayers were dried over MgSO₄ and concentrated to give the desiredproduct 3a as a white solid (132 mg, 95%). ¹H NMR (300 MHz, D₂O) δ 4.96(d, 1H, J=3.0 Hz), 3.07 (m, 1H), 2.04 (s, 3H), 1.15 (d, 3H, J=7.2 Hz);¹⁹F NMR (282.6 MHz, D₂O/CF₃CO₂H) δ −71.63 (d, 3F, J=8.8 Hz); FT-IR (KBrpellet, v_(max), cm⁻¹) 3397s (br), 3253s, 3068m, 2981s, 2948m, 1686s,1552s, 1369s, 1289s, 1174s, 1145s, 1055s; GC-MS (CI, CH₄): 214 (100,[M+1]⁺), 196 (9), 172 (33), 82 (33), 57 (6).

(2S,3R)-, (2R,3S)-N-Ac-4,4,4-trifluorovaline (3b)

¹H NMR (300 MHz, D₂O) δ 4.67 (d, 1H, J=3.3 Hz), 3.07 (m, 1H), 2.04 (s,3H), 1.17 (d, 3H, J=7.2 Hz); ¹⁹F NMR (282.6 MHz, D₂O/CF₃CO₂H) δ −69.43(d, 3F, J=8.8 Hz); FT-IR (KBr pellet, v_(max), cm⁻¹) 3397s (br), 3253s,3068m, 2981s, 2948m, 1686s, 1552s, 1369s, 1289s, 1174s, 1145s, 1055s;GC-MS (CI, CH₄): 214 (100, [M+1]⁺), 196 (9), 172 (33), 101 (10), 82(33), 57 (6).

EXAMPLE 15

(2S,3S)-4,4,4-Trifluorovaline (4a)

To a solution of 3a (107 mg, 0.5 mmol) in 1 mL of pH 7.9 aq. LiOH/HOAcwas added porcine kidney acylase I (10 mg) at 25° C. The mixture wasstirred at 25° C. for 48 h (pH was maintained at 7.5 by periodicaddition of 1 N LiOH). The reaction was then diluted with 5 mL of water,acidified to pH 5.0, heated to 60° C. with Norit, and filtered. Thefiltrate was acidified to pH 1.5 and extracted with ethyl acetate (2×10mL). The aqueous layer was freeze-dried to give 49 mg of 4a (95%). Thecombined organic layers were concentrated, and the residue refluxed in 3N HCl for 6 h, then freeze-dried to yield 50 mg of 4c (98%).

The other two diastereomers, 4b and 4d, were obtained from 3b using thesame procedure.

(2S,3S)-4,4,4-Trifluorovaline (4a)

¹H NMR (300 MHz, D₂O) δ 4.24 (dd, 1H, J=2.1, 3.9 Hz), 3.23 (m, 1H), 1.30(d, 3H, J=7.2 Hz); ¹⁹F NMR (282.6 MHz, D₂O/CF₃CO₂H) δ −71.69 (d, 3F,J=9.3 Hz); [α]_(D) ^(23.7)=+7.2° (c 0.75, 1 N HCl).

(2S,3R)-4,4,4-Trifluorovaline (4b)

¹H NMR (300 MHz, D₂O) δ 4.35 (t, 1H, J=2.7 Hz), 3.27 (m, 1H), 1.22 (d,3H, J=7.5 Hz); ¹⁹F NMR (282.6 MHz, D₂O/CF₃CO₂H) δ −70.04 (d, 3F, J=9.0Hz); [α]_(D) ^(23.3)=+12.8° (c 0.5, 1 N HCl).

(2R,3R)-4,4,4-Trifluorovaline (4c)

¹H NMR (300 MHz, D₂O) δ 4.24 (dd, 1H, J=2.1, 3.9 Hz), 3.23 (m, 1H), 1.30(d, 3H, J=7.2 Hz); ¹⁹F NMR (282.6 MHz, D₂O/CF₃CO₂H) δ −70.04 (d, 3F,J=9.0 Hz).

(2R,3S)-4,4,4-Trifluorovaline (4d)

¹H NMR (300 MHz, D₂O) δ 4.35 (t, 1H, J=2.7 Hz), 3.27 (m, 1H), 1.22 (d,3H, J=7.5 Hz); ¹⁹F NMR (282.6 MHz, D₂O/CF₃CO₂H) δ −71.69 (d, 3F, J=9.3Hz).

EXAMPLE 16

N-Boc-5,5,5-trifluoroleucine methyl ester (6)

A mixture of Boc-DL-trifluoroleucine (1.25 g, 4.38 mmol), iodomethane(0.3 mL, 4.82 mmol), NaHCO₃ (1.1 g, 13.15 mmol), and dry DMF (20 mL) wasstirred at room temperature under argon for 6 h, then diluted with 200mL of ethyl acetate, and washed with water (4×100 mL). The organic layerwas dried over Na₂SO₄ and concentrated to give 1.25 g of product as apale-yellow oil (95%). Column chromatography on silica gel (500 g) usingEt₂O/n-pentane (1:4) as eluant afforded 420 mg of (2S,4R)-,(2R,4S)-N-Boc-5,5,5-trifluoroleucine methyl ester (6a) (32%), 347 mg of(2S,4S)-, (2R,4R)-N-Boc-5,5,5-trifluoroleucine methyl ester (6b) (27%),and 337 mg of the mixture of 6a and 6b (26%).

(2S,4R)-, (2R,4S)-N-Boc-5,5,5-trifluoroleucine methyl ester (6a)

¹H NMR (300 MHz, CDCl₃) δ 5.29 (d, 1H, J=6.9 Hz), 4.32 (m, 1H), 3.70 (s,3H), 2.31 (m, 1H), 2.12 (m, 1H), 1.58 (m, 1H), 1.37 (s, 9H), 1.11 (d,3H, J=6.9 Hz); ¹³C NMR (75.5 MHz, CDCl₃) δ 172.72 (C═O), 155.29 (C═O),128.09 (q, CF₃, ¹J_(CF)=278.9 Hz), 80.27 (C), 52.54 (CH₃), 51.70 (CH),35.13 (q, CH, ²J_(CF)=26.4 Hz), 32.98 (CH₂), 28.30 (3×CH₃), 13.17 (CH₃);¹⁹F NMR (282.6 MHz, CDCl₃/CFCl₃) δ −74.15 (d, 3F, J=8.2 Hz); FT-IR(film, v_(max) cm⁻¹) 3360m, 2984m, 2938m, 1747s, 1716s, 1520s, 1368s,1269s, 1168s, 1133m; GC-MS (CI, CH₄): 300 (2, [M+1]⁺), 284 (7), 244(100), 200 (66), 82 (21), 57 (24).

(2S,4S)-, (2R,4R)-N-Boc-5,5,5-trifluoroleucine methyl ester (6b)

¹H NMR (300 MHz, CDCl₃) δ 5.02 (d, 1H, J=8.7 Hz), 4.38 (m, 1H), 3.76 (s,3H), 2.32 (m, 1H), 1.91–1.74 (br. m, 2H), 1.44 (s, 9H), 1.20 (d, 3H,J=6.9 Hz); ¹³C NMR (75.5 MHz, CDCl₃) δ 173.03 (C═O), 155.86 (C═O),128.24 (q, CF₃, ¹J_(CF)=278.9 Hz), 80.57 (C), 52.80 (CH₃), 50.83 (CH),35.02 (q, CH, ²J_(CF)=26.9 Hz), 33.00 (CH₂), 28.42 (3×CH₃), 12.28 (CH₃);¹⁹F NMR (282.6 MHz, CDCl₃/CFCl₃) δ −74.03 (d, 3F, J=8.7 Hz); FT-IR (KBrpellet, v_(max), cm⁻¹) 3368s, 3014m, 2983s, 2961m, 1763s, 1686s, 1527s,1265s, 1214s, 1170s, 1053s, 1028s; GC-MS (CI, CH₄): 300 (2, [M+1]⁺), 284(7), 244 (100), 224 (30), 200 (66), 57 (24).

EXAMPLE 17

(2S,4R)-, (2R,4S)-N-Ac-5,5,5-trifluoroleucine (7a)

(2S,4R)-, (2R,4S)-N-Boc-5,5,5-trifluoroleucinol

To a solution of 6a (420 mg, 1.4 mmol) in methanol (10 mL) was addedNaBH₄ (531 mg, 14.0 mmol) in small portions. The reaction mixture wasstirred at room temperature for 1 h before removal of the solvent. Theresidue was partitioned between 100 mL of ethyl acetate and 50 mL ofwater. The aqueous layer was extracted with 100 mL of ethyl acetate. Thecombined organic layers were dried over Na₂SO₄ and concentrated to yield357 mg of the desired product as a white solid (94%). ¹H NMR (300 MHz,CDCl₃) δ 4.74 (m, 1H), 3.71 (m, 2H), 3.58 (m, 1H), 2.31 (m, 1H), 2.14(m, 1H), 1.92 (m, 1H), 14.5 (s, 9H), 1.17 (d, 3H, J=7.0 Hz). ¹³C NMR(75.5 MHz, CDCl₃) δ 156.26 (C═O), 128.41 (q, CF₃, ¹J_(CF)=279.4 Hz),80.14 (C), 64.78 (CH₂), 50.73 (CH), 35.59 (q, CH, ²J_(CF)=29.6 Hz),31.74 (CH₂), 28.52 (3×CH₃), 13.71 (CH₃); ¹⁹F NMR (282.6 MHz,CDCl₃/CFCl₃)δ −73.84 (br. s, 3F); GC-MS (CI, CH₄): 272 (100, [M+1]⁺),216 (68), 172 (26), 57 (11).

(2S,4S)-, (2R,4R)-N-Boc-5,5,5-trifluoroleucinol

¹H NMR (300 MHz, CDCl₃) δ 4.58 (m, 1H), 3.79 (m, 1H), 3.68 (m, 1H), 3.58(m, 1H), 2.27 (m, 1H), 2.05 (m, 1H), 1.80 (m, 1H), 1.45 (s, 9H), 1.18(d, 3H, J=6.6 Hz). ¹³C NMR (75.5 MHz, CDCl₃) δ 156.47 (C═O), 128.56 (q,CF₃, ¹J_(CF)=278.7 Hz), 80.20 (C), 66.31 (CH₂), 49.49 (CH), 35.15 (q,CH, ²J_(CF)=26.7 Hz), 31.71 (CH₂), 28.50 (3×CH₃), 12.56 (CH₃); ¹⁹F NMR(282.6 MHz, CDCl₃/CFCl₃) δ −73.98 (d, 3F, J=8.5 Hz); GC-MS (CI, CH₄):272 (100, [M+1]⁺), 172 (26), 57 (11).

(2S,4R)-, (2R,4S)-N-Ac-5,5,5-trifluoroleucine (7a)

A mixture of (2S,4R)-, (2R,4S)-N-Boc-5,5,5-trifluoroleucinol (330 mg,1.23 mmol), PDC (4.62 g, 12.3 mmol), and dry DMF (2.5 mL) was stirred atroom temperature under argon for 4 h, then diluted with 50 mL of ethylacetate and 50 mL of water. The organic layer was washed with 30 mL of1N HCl and 2×30 mL of water, dried over MgSO₄, and concentrated to give198 mg of (2S,4R)-, (2R,4S)-N-Boc-5,5,5-trifluoroleucine as apale-brownish oil (60%).

A solution of the above product (180 mg, 0.63 mmol) in 2 mL of CH₂Cl₂was treated with 0.5 mL of trifluoroacetic acid for 30 min at roomtemperature. After removal of the solvent, the yellowish residue wasdissolved in 2 mL of water, treated with NaOH (126 mg, 3.15) at 0° C.,and acetic anhydride (0.12 mL, 1.26 mmol) was then added dropwise. Thereaction mixture was stirred at 0° C. for 30 min, then allowed to warmto room temperature. After stirring for another 1 h, the mixture wasdiluted with 30 mL of water, acidified to pH 2 with 3 N HCl, andextracted with ethyl acetate (2×90 mL). The combined organic layers weredried over Na₂SO₄ and concentrated to yield 136 mg of 7a as a whitesolid (95%). ¹H NMR (300 MHz, D₂O) δ 4.48 (dd, 1H, J=6.1, 8.8 Hz), 2.51(m, 1H), 2.27 (m, 1H), 2.06 (s, 3H), 1.79 (m, 1H), 1.18 (d, 3H, J=7.0Hz); ¹³C NMR (75.5 MHz, D₂O) δ 175.48 (C═O), 174.60 (C═O), 128.53 (q,CF₃, ¹J_(CF)=278.9 Hz), 51.24 (CH), 34.88 (q, CH, ²J_(CF)=26.6 Hz),31.21 (CH₂), 21.90 (CH₃), 13.03 (CH₃); ¹⁹F NMR (282.6 MHz, D₂O/CF₃CO₂H)δ −73.68 (d, 3F, J=9.0 Hz); FT-IR (KBr pellet, v_(max), cm⁻¹) 3343s,3063–2487m (br.), 2932m, 2894m, 1709s, 1613s, 1549s, 1266s, 1179s,1137s; GC-MS (CI, CH₄): 228 (100, [M+1]⁺), 211 (47), 186 (26), 140 (16),57 (11).

(2S,4S)-, (2R,4R)-N-Ac-5,5,5-trifluoroleucine (7b)

¹H NMR (300 MHz, D₂O) δ 4.48 (dd, 1H, J=3.8, 11.6 Hz), 2.41 (m, 1H),2.07 (s, 3H), 2.15–1.91 (br. m, 2H), 1.16 (d, 3H, J=6.9 Hz); ¹³C NMR(75.5 MHz, D₂O) δ 178.35 (C═O), 177.38 (C═O), 131.09 (q, CF₃,¹J_(CF)=278.3 Hz), 52.72 (CH), 37.31 (q, CH, ²J_(CF)=26.6 Hz), 33.06(CH₂), 24.50 (CH₃), 13.90 (CH₃); ¹⁹F NMR (282.6 MHz, D₂O/CF₃CO₂H) δ−73.87 (d, 3F, J=8.5 Hz); FT-IR (KBr pellet, v_(max), cm⁻¹) 3336s,2977m, 2949m, 2897m, 2615m, 2473s, 1711 s, 1628s, 1551s, 1276s, 1250s,1127s, 1095s; GC-MS (CI, CH₄): 228 (100, [M+1]⁺), 211 (47), 186 (26),140 (16), 120 (3), 57 (11).

EXAMPLE 18

(2S,4R)-5,5,5-Trifluoroleucine (8a)

To a solution of 7a (136 mg, 0.6 mmol) in 2 mL of pH 7.9 aqueousLiOH/HOAc was added porcine kidney acylase I (18 mg) at 27° C. Themixture was stirred at 27° C. for 48 h (pH was maintained at 7.5 byperiodic addition of 1 N LiOH). It was further diluted with 5 mL ofwater, acidified to pH 5.0, heated to 60° C. with Norit, and filtered.The filtrate was acidified to pH 1.5 and extracted with ethyl acetate(2×50 mL). The aqueous layer was freeze-dried to give 63 mg of 8a (95%).The combined organic layers were concentrated, and the residue refluxedin 3 N HCl for 6 h, then freeze-dried to yield 64 mg of 8c (96%).

The other two diastereomers, 8b and 8d, were obtained from 7b using thesame procedure.

(2S,4R)-5,5,5-Trifluoroleucine (8a)

¹⁹F NMR (282.6 MHz, D₂O/CF₃CO₂H) δ −74.33 (d, 3F, J=9.0 Hz); [α]_(D)^(22.9)=+21.6° (c 0.5, 1N HCl).

(2S,4S)-5,5,5-Trifluoroleucine (8b)

¹⁹F NMR (282.6 MHz, D₂O/CF₃CO₂H) δ −74.11 (d, 3F, J=8.2 Hz); [α]_(D)^(23.6)=−4.0° (c 0.8, 1N HCl).

(2R,4S)-5,5,5-Trifluoroleucine (8c)

¹⁹F NMR (282.6 MHz, D₂O/CF₃CO₂H) δ −74.33 (d, 3F, J=9.0 Hz).

(2R,4R)-5,5,5-Trifluoroleucine (8d)

¹⁹F NMR (282.6 MHz, D₂O/CF₃CO₂H) δ −74.11 (d, 3F, J=8.2 Hz).

EXAMPLE 19

Boc-TFV(2S,3S)-Ser(Ot-Bu)-OMe(2S)

To a stirred solution of (2S,4S)-5,5,5-Trifluorovaline (4b) (5 mg, 0.02mmol) in DMF (1 mL) was added diisopropylethyl amine (DIEA, 0.01 mL,0.06 mmol), O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU, 8 mg, 0.02 mmol), and the HCl salt of(2S)-H-Ser(Ot-Bu)-OMe (9 mg, 0.04 mmol), sequentially. The mixture wasstirred at room temperature for 20 min before dilution with water (5 mL)and extraction with diethyl ether (15 mL). The organic layer was washedwith 1 N HCl (2×5 mL) and 5% NaHCO₃ (2×8 mL), dried over MgSO₄, andconcentrated to give 7 mg of the dipeptide (88%). ¹H NMR (300 MHz,CDCl₃) δ 6.92 (d, 1H, J=7.8 Hz), 5.16 (d, 1H, J=8.7 Hz), 4.65 (m, 1H),4.39 (dd, 1H, J=5.1, 8.8 Hz), 3.81 (dd, 1H, J=2.7, 9.0 Hz), 3.74 (s,3H), 3.56 (dd, 1H, J=3.0, 9.0 Hz), 3.04 (m, 1H), 1.46 (s, 9H), 1.23 (d,3H, J=7.2 Hz), 1.14 (s, 9H); ¹⁹F NMR (282.6 MHz, CDCl₃/CFCl₃) δ −68.57(d, 3F, J=8.7 Hz).

Boc-TFV(2S,3R)-Ser(Ot-Bu)-OMe(2S)

¹⁹F NMR (282.6 MHz, CDCl₃/CFCl₃) δ −71.36 (d, 3F, J=7.9 Hz).

Boc-TFV(2R,3S)-Ser(Ot-Bu)-OMe(2S)

¹⁹F NMR (282.6 MHz, CDCl₃/CFCl₃) δ −71.48 (d, 3F, J=8.5 Hz).

Boc-TFV(2R,3R)-Ser(Ot-Bu)-OMe(2S)

¹⁹F NMR (282.6 MHz, CDCl₃/CFCl₃) δ −68.49 (d, 3F, J=9.0 Hz).

Incorporation by Reference

All of the patents and publications cited herein are hereby incorporatedby reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A compound represented by A:

wherein X represents O, S, N(R²), or C(R³)₂; R represents independentlyfor each occurrence H, alkyl, aryl, heteroaryl, heteroaralkyl, formyl,acyl, alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl; R² represents independently for each occurrence H,alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, formyl, acyl,alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl; R³ represents independently for each occurrence H,alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, formyl, acyl,alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl; R′ represents H, alkyl, aryl, heteroaryl, aralkyl,or heteroaralkyl; or XR′ represents halide; the stereochemicalconfiguration at any stereocenter of a compound represented by A may beR, S, or a mixture of these configurations; and the enantiomeric excessof a compound represented by A is greater than or equal to about 95%. 2.The compound of claim 1, wherein X represents O or N(R²).
 3. Thecompound of claim 1, wherein R represents independently for eachoccurrence H, alkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; R² represents independentlyfor each occurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl,aralkoxycarbonyl, or aralkylaminocarbonyl; and R³ representsindependently for each occurrence H, alkyl, aralkyl, acyl,alkoxycarbonyl, aralkoxycarbonyl, or aralkylaminocarbonyl.
 4. Thecompound of claim 1, wherein R, R² and R³ represent independently foreach occurrence H.
 5. The compound of claim 1, wherein R′ represents H,alkyl, or aralkyl.
 6. The compound of claim 1, wherein R′ represents H.7. The compound of claim 1, wherein R, R² and R³ represent independentlyfor each occurrence H; and R′ represents H.
 8. The compound of claim 1,wherein X represents O or N(R²); R represents independently for eachoccurrence H, alkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; R² represents independentlyfor each occurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl,aralkoxycarbonyl, alkylaminocarbonyl, or aralkylaminocarbonyl; and R³represents independently for each occurrence H, alkyl, aralkyl, acyl,alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl.
 9. The compound of claim 1, wherein X represents Oor N(R²); and R and R² represent independently for each occurrence H.10. The compound of claim 1, wherein X represents O or N(R²); and R′represents H, alkyl, or aralkyl.
 11. The compound of claim 1, wherein Xrepresents O or N(R²); and R′ represents H.
 12. The compound of claim 1,wherein X represents O or N(R²); R represents independently for eachoccurrence H, alkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; R² represents independentlyfor each occurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl,aralkoxycarbonyl, alkylaminocarbonyl, or aralkylaminocarbonyl; and R′represents H, alkyl, or aralkyl.
 13. The compound of claim 1, wherein Xrepresents O or N(R²); R, and R² represent independently for eachoccurrence H; and R′ represents H, alkyl, or aralkyl.
 14. The compoundof claim 1, wherein X represents O or N(R²); R represents independentlyfor each occurrence H, alkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; R² represents independentlyfor each occurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl,aralkoxycarbonyl, alkylaminocarbonyl, or aralkylaminocarbonyl; and R′represents H.
 15. The compound of claim 1, wherein X represents 0 orN(R²); R, and R² represent independently for each occurrence H; and R′represents H.
 16. The compound of claim 1, wherein said compound is asingle stereoisomer.
 17. The compound of claim 1, wherein said compoundis in the form of a salt.
 18. A formulation, comprising a compound ofclaim 1 and a pharmaceutically acceptable excipient.
 19. The compound ofclaim 2, wherein said compound is a single stereoisomer.
 20. Thecompound of claim 3, wherein said compound is a single stereoisomer. 21.The compound of claim 4, wherein said compound is a single stereoisomer.22. The compound of claim 5, wherein said compound is a singlestereoisomer.
 23. The compound of claim 6, wherein said compound is asingle stereoisomer.
 24. The compound of claim 7, wherein said compoundis a single stereoisomer.
 25. The compound of claim 8, wherein saidcompound is a single stereoisomer.
 26. The compound of claim 9, whereinsaid compound is a single stereoisomer.
 27. The compound of claim 10,wherein said compound is a single stereoisomer.
 28. The compound ofclaim 11, wherein said compound is a single stereoisomer.
 29. Thecompound of claim 12, wherein said compound is a single stereoisomer.30. The compound of claim 13, wherein said compound is a singlestereoisomer.
 31. The compound of claim 14, wherein said compound is asingle stereoisomer.
 32. The compound of claim 15, wherein said compoundis a single stereoisomer.